Note: Descriptions are shown in the official language in which they were submitted.
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IMPROVED IMAGING AGENTS COMPRISING BARBITURIC ACID DERIVATIVES
Field of the Invention.
The present invention relates to diagnostic imaging agents for in vivo
imaging. The
imaging agents comprise a synthetic barbituric acid derivative labelled at the
5-position
with an imaging moiety suitable for diagnostic imaging in vivo.
Ba~ound to the Invention.
Barbituric acid, or pyrimidine-2,4,6-trione is a known drug. Derivatives
thereof,
O
HN- _NH
O'~\~O
Barbituric acid
especially those arising from the introduction of substituents at the 5-
position (ie. the CH2
of the pyrimidine ring) are also known drugs. An example is barbital, ie. 5,5-
diethylbarbituric acid.
Grigsby et al [J.Nucl.Med., 22(6), Abstract P12 (1981)] disclose lipophilic
~SSe and
iasmTe-labelled barbiturate derivatives, where the radioisotope is part of an
aralkyl
substituent at the S-position, as potential regional cerebral blood flow
imaging
radiopharmaceuticals.
US 3952091 discloses compounds useful in the in vitro radioimmunoassay of
barbiturate
drugs, which comprise barbituric acid labelled at the 5-position with the
radioisotope lzsl.
US 4244939 discloses compounds useful in the in vitro radioimmunoassay of
barbiturate
drugs, which comprise barbituric acid labelled at 1- or 3- position (ie. the
ring nitrogens),
optionally via a linker group, with the radioisotopes Iasl or l3il.
CONFIRMATION COPY
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WO 01/60416 discloses chelator conjugates of matrix metalloproteinase (MMP)
inhibitors, and their use in the preparation of metal complexes with
diagnostic metals.
The specific classes of MMP inhibitor described are hydroxamates, especially
succinyl
hydroxamates.
The Present Invention.
It has now been found that synthetic barbituric acid matrix metalloproteinase
(MMP)
inhibitors labelled at the 5-position with an imaging moiety are useful
diagnostic imaging
agents for in vivo imaging of the mammalian body. Barbituric acid MMP
inhibitors (ie.
pyrimidine-2,4,6-triones) can exhibit greater selectivity than hydroxamic acid
derivatives
for selected MMPs, especially for the gelatinases (MMP-2 and MMP-9), and the
membrane-bound MT-MMPs 1 (MMP-14) and 3 (MMP-16), plus MMP-8. For an
imaging agent this results in decreased unwanted background activity, and
hence
improved signal to noise. Barbituric acid derivatives are also more lipophilic
than
hydroxamic acid or peptide-based MMP inhibitors, which means that the imaging
agents
of the present invention are better able to cross cell membranes or the blood-
brain barrier
due to their lipophilicity. Hence, the agents of the present invention are
expected to be
useful also for imaging brain disease such as brain tumours, amyotrophic
lateral sclerosis,
2o Alzheimer's disease or other sites of MMP activity within the brain.
The imaging agents of the present invention are useful for the in vivo
diagnostic imaging
of a range of disease states (inflammatory, malignant and degenerative
diseases) where
specific matrix metalloproteinases are known to be involved. These include:
(a) atherosclerosis, where various MMPs are overexpressed. Elevated levels of
MMP-1, 3, 7, 9, 1 l, 12, 13 and MTl-MMP have been detected in human
atherosclerotic plaques [S.J. George, Exp. Opin. Invest. Drugs, 9(S), 993-1007
(2000) and references therein]. Expression of MMP-2 [Z. Li et al, Am. J.
Pathol.,
148, 121-128 (1996)] and MMP-8 [M. P. Herman et al, Circulation, 104, 1899-
1904 (2001)] in human atheroma has also been reported;
(b) chronic heart failure (Peterson, J. T. et al. Matrix metalloproteinase
inhibitor
development for the treatment of heart failure, Drug Dev. Res. (2002), 55(1),
29-
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WO 2004/032936 PCT/GB2003/004351
44 reports that MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-13 and MMP-
14 are upregulated in heart failure);
(c) cancer [Vihinen et al, Int. J. Cancer 99, p157-166 (2002) reviews MMP
involvement in cancers, and particularly highlights MMP-2, MMP-3, MMP-7, and
s MMP-9];
(d) arthritis [Jacson et al, Inflamm. Res. SO(4), p183-186 (2001) "Selective
matrix
metalloproteinase inhibition in rheumatoid arthritis - targeting gelatinase A
activation", MMP-2 is particularly discussed);
(e) amyotrophic lateral sclerosis [Lim et al, J.Neurochem, 67, 251-259 (1996);
where MMP-2 and MMP-9 are involved];
(f) brain metastases, where MMP-2, MMP-9 and MMP-13 have been reported to
be implicated [Spinale, Circul.Res., 90, 520-530 (2002)];
(g) cerebrovascular diseases, where MMP-2 and MMP-9 have been reported to be
involved [Lukes et al, Mol.Neurobiol., 19, 267-284 (1999)];
1 s (h) Alzheimer's disease, where MMP-2 and MMP-9 have been identified in
diseased tissue [Backstrom et al, J.Neurochem., 58, 983-992 (1992)];
(i) neuroinflammatory disease, where MMP-2, MMP-3 and MMP-9 are involved
[Mun-Bryce et al, Brain.Res., 933, 42-49 (2002)];
(j) COPD (ie. chronic obstructive pulmonary disease) where MMP-1, MMP-2,
2o MMP-8 and MMP-9 have been reported to be upregulated [Segura-Valdez et al,
Chest, 117, 684-694 (2000)];
(k) eye pathology [Kurpakus-Wheater et al, Prog. Histo. Cytochem., 36(3), 179-
259 (2001)];
(1) skin diseases [Herouy, Y., Int. J. Mol. Med., 7(1), 3-12 (2001)].
2s
Detailed Description of the Invention.
In a first aspect, the present invention provides an imaging agent which
comprises a
synthetic barbituric acid matrix metalloproteinase inhibitor labelled at the S-
position of
3o the barbituric acid with an imaging moiety, wherein the imaging moiety can
be detected
following administration of said labelled synthetic barbituric acid matrix
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4
metalloproteinase inhibitor to the mammalian body in vivo, and said imaging
moiety is
chosen from:
(i) a radioactive metal ion;
(ii) a paramagnetic metal ion;
(iii) a gamma-emitting radioactive halogen;
(iv) a positron-emitting radioactive non-metal;
(v) a hyperpolarised NMR-active nucleus;
(vi) a reporter suitable for in vivo optical imaging;
(vii) a [3-emitter suitable for intravascular detection.
The synthetic barbituric acid matrix rnetalloproteinase inhibitor is suitably
of molecular
weight 100 to 2000 Daltons, preferably of molecular weight 150 to 600 Daltons,
and
most preferably of molecular weight 200 to 500 Daltons.
The imaging moiety may be detected either external to the mammalian body or
via use of
detectors designed for use in vivo, such as intravascular radiation or optical
detectors such
as endoscopes, or radiation detectors designed for infra-operative use.
Preferred imaging
moieties are those which can be detected externally in a non-invasive manner
following
administration in vivo. Most preferred imaging moieties are radioactive,
especially
radioactive metal ions, gamma-emitting radioactive halogens and positron-
emitting
radioactive non-metals, particularly those suitable for imaging using SPECT or
PET.
When the imaging moiety is a radioactive metal ion, ie. a radiometal, suitable
radiometals
can be either positron emitters such as 64Cu; 48V, saFe, ssCo, 94mTc or 68Ga;
y-emitters
such aS 99mTC, I I iln, i lsmln, or 6~Ga. Preferred radiometals are 99"'Tc,
64Cu, 68Ga and
i llIn. Most preferred radiometals are y-emitters, especially 99"1TC.
When the imaging moiety is a paramagnetic metal ion, suitable such metal ions
include:
Gd(III), Mn(II), Cu(II), Cr(III), Fe(III), Co(II), Er(II), Ni(II), Eu(III) or
Dy(III). Preferred
paramagnetic metal ions are Gd(III), Mn(II) and Fe(III), with Gd(III) being
especially
preferred.
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When the imaging moiety is a gamma-emitting radioactive halogen, the
radiohalogen is
suitably chosen from lz3h isy or ~~Br. A preferred gamma-emitting radioactive
halogen
is i23I.
5 When the imaging moiety is a positron-emitting radioactive non-metal,
suitable such
positron emitters include: 1'C, 13N, is0, mF, isF, ~sBr, ~6Br or laaI.
preferred positron-
emitting radioactive non-metals are 11C, i3N and 18F, especially i1C and 18F,
most
especially 18F.
When the imaging moiety is a hyperpolarised NMR-active nucleus, such NMR-
active
nuclei have a non-zero nuclear spin, and include 13C, lsN, 19F, ~9Si and 31P.
Of these, 13C
is preferred. By the term "hyperpolarised" is meant enhancement of the degree
of
polarisation of the NMR-active nucleus over its' equilibrium polarisation. The
natural
abundance of 13C (relative to laC) is about 1%, and suitable 13C-labelled
compounds are
suitably enriched to an abundance of at least 5%, preferably at least 50%,
most preferably
at least 90% before being hyperpolarised. At least one carbon atom of a carbon-
containing substituent at the 5-position of the barbituric acid of the present
invention is
suitably enriched with'3C, which is subsequently hyperpolarised.
When the imaging moiety is a reporter suitable for in vivo optical imaging,
the reporter is
any moiety capable of detection either directly or indirectly in an optical
imaging
procedure. The reporter might be a light scatterer (eg. a coloured or
uncoloured particle),
a light absorber or a light emitter. More preferably the reporter is a dye
such as a
chromophore or a fluorescent compound. The dye can be any dye that interacts
with light
in the electromagnetic spectrum with wavelengths from the ultraviolet light to
the near
infrared. Most preferably the reporter has fluorescent properties.
Preferred organic chromophoric and fluorophoric reporters include groups
having an
extensive delocalized electron system, eg. cyanines, merocyanines,
indocyanines,
phthalocyanines, naphthalocyanines, triphenylmethines, porphyrins, pyrilium
dyes,
thiapyriliup dyes, squarylium dyes, croconium dyes, azulenium dyes,
indoanilines,
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benzophenoxazinium dyes, benzothiaphenothiazinium dyes, anthraquinones,
napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes,
intramolecular and intermolecular charge-transfer dyes and dye complexes,
tropones,
tetrazines, bis(dithiolene) complexes, bis(benzene-dithiolate) complexes,
iodoaniline
dyes, bis(S,O-dithiolene) complexes. Fluorescent proteins, such as green
fluorescent
protein (GFP) and modifications of GFP that have different absorption/emission
properties are also useful. Complexes of certain rare earth metals (e.g.,
europium,
samarium, terbium or dysprosium) are used in certain contexts, as are
fluorescent
nanocrystals (quantum dots).
to
Particular examples of chromophores which may be used include: fluorescein,
sulforhodamine 101 (Texas Red), rhodamine B, rhodamine 6G, rhodarnine 19,
indocyanine green, Cy2, Cy3, Cy3.5, CyS, Cy5.5, Cy7, Marina Blue, Pacific
Blue,
Oregon Green 48$, Oregon Green 514, tetramethylrhodamine, and Alexa Fluor 350,
15 Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa
Fluor 568,
Alexa F'luor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa
Fluor 680,
Alexa Fluor 700, and Alexa Fluor 750.
Particularly preferred are dyes which have absorption maxima in the visible or
near
2o infrared region, between 400 nm and 3 Vim, particularly between 600 and
1300 nm.
Optical imaging modalities and measurement techniques include, but not limited
to:
luminescence imaging; endoscopy; fluorescence endoscopy; optical coherence
tomography; transmittance imaging; time resolved transmittance imaging;
confocal
25 imaging; nonlinear microscopy; photoacoustic imaging; acousto-optical
imaging;
spectroscopy; reflectance spectroscopy; interferometry; coherence
interferometry; diffuse
optical tomography and fluorescence mediated diffuse optical tomography
(continuous
wave, time domain and frequency domain systems), and measurement of light
scattering,
absorption, polarisation, luminescence, fluorescence lifetime, quantum yield,
and
30 quenching.
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7
When the imaging moiety is a /3-emitter suitable for intravascular detection,
suitable such
(3-emitters include the radiometals 6~Cu, $9Sr, 9~Y, ls3Sm, IasRe, issRe or
l9zh., and the
non-metals 32P, 33P, ssS~ ssCh 39Ch s2Br and s3Br.
The imaging agents of the present invention are preferably of Formula I:
[{inhibitor]-(A)n]m [imaging moiety] (I)
where:
{inhibitor} is the synthetic barbituric acid matrix metalloproteinase
inhibitor;
-(A)n is a linker group wherein each A is independently -CR2- , -CR=CR- ,
-C---C- , -CRZC02- , -C02CR~- , NRCO- , -CONR- , -NR(C=O)NR-,
-NR(C=S)NR-, -SOZNR- , -NRSOZ- , -CR2OCR2- , -CR2SCR2- , -CR2NRCR2- , a
C4_s cycloheteroalkylene group, a C4_s cycloalkylene group, a Cs_IZ arylene
group,
or a C3_i2 heteroarylene group, an amino acid or a monodisperse
polyethyleneglycol (PEG) building block;
where R is independently chosen from H, Cl~ alkyl, C2~ alkenyl,
Cap alkynyl, Cl~ alkoxyalkyl or C» hydroxyalkyl;
n is an integer of value 0 to 10, and
m is l, 2 or 3.
It is envisaged that the role of the linker group -(A)n of Formula I is to
distance the
imaging moiety from the active site of the barbiturate metalloproteinase
inhibitor. This is
particularly important when the imaging moiety is relatively bulky (eg. a
metal complex),
so that binding of the inhibitor to the MMP enzyme is not impaired. This can
be achieved
by a combination of flexibility (eg. simple alkyl chains), so that the bulky
group has the
freedom to position itself away from the active site and/or rigidity such as a
cycloalkyl or
aryl spacer which orientates the metal complex away from the active site.
The nature of the linker group can also be used to modify the biodistribution
of the
imaging agent. Thus, eg. the introduction of ether groups. in the linker will
help to
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minimise plasma protein binding. When -(A}n comprises a monodisperse
polyethyleneglycol (PEG) building block or a peptide chain of 1 to 10 amino
acid
residues, the linker group may function to modify the pharmacokinetics and
blood
clearance rates of the imaging agent in vivo. Such "biomodifier" linker groups
may
accelerate the clearance of the imaging agent from background tissue, such as
muscle or
liver, and/or from the blood, thus giving a better diagnostic image due to
less background
interference. A biomodifier linker group may also be used to favour a
particular route of
excretion, eg. via the kidneys as opposed to via the liver.
When -(A)" comprises a peptide chain of 1 to 10 amino acid residues, the amino
acid
residues are preferably chosen from glycine, lysine, aspartic acid or serine.
When -(A)"
comprises a PEG moiety, it preferably comprises a unit derived from
polymerisation of
the monodisperse PEG-like structure, 17-amino-5-oxo-6-aza-3, 9, 12, 15-
tetraoxaheptadecanoic acid of Formula II:
H
~HN~O~O~O~/N O
O ~ ~~-~~JnO
is (II)
wherein n equals an integer from 1 to 10 and where the C-terminal unit (*) is
connected
to the imaging moiety.
When the linker group does not comprise PEG or a peptide chain, preferred -
(A)" groups
2o have a backbone chain of linked atoms which make up the -(A)n moiety of 2
to 10 atoms,
most preferably 2 to 5 atoms, with 2 or 3 atoms being especially preferred. A
minimum
linker group backbone chain of 2 atoms confers the advantage that the imaging
moiety is
well-separated from the barbituric acid metalloproteinase inhibitor so that
any interaction
is minimised.
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9
Non-peptide linker groups such as alkylene groups or arylene groups have the
advantage
that there are no significant hydrogen bonding interactions with the
conjugated barbituric
acid MMP inhibitor, so that the linker does not wrap round onto the barbituric
acid MMP
inhibitor. Preferred alkylene spacer groups are -(CHZ)q where q is 2 to 5.
Preferred
arylene spacers are of formula:
-(CH2)a ~ ~ (CH2)b
where: a and b are independently 0, 1 or 2.
The linker group -(A)n is preferably derived from glutaric acid, succinic
acid, a
polyethyleneglycol based unit or a PEG-like unit of Formula II.
When the imaging moiety comprises a metal ion, the metal ion is present as a
metal
complex. Such barbituric acid metalloproteinase inhibitor conjugates with
metal ions are
therefore suitably of Formula Ia:
[{inhibitor}-(A)"Jm [metal complex] (Ia)
where: A, n and m are as defined for Formula I above.
By the term "metal complex" is meant a coordination complex of the metal ion
with one
or more ligands. It is strongly preferred that the metal complex is "resistant
to
transchelation", ie. does not readily undergo ligand exchange with other
potentially
competing ligands for the metal coordination sites. Potentially competing
ligands include
the barbituric acid moiety itself plus other excipients in the preparation in
vitro (eg.
radioprotectants or antimicrobial preservatives used in the preparation), or
endogenous
compounds in vivo (eg. glutathione, transfernn or plasma proteins).
The metal complexes of Formula I are derived from conjugates of ligands of
Formula Ib:
[ f inhibitor}-(A)"]m [ligand] (Ib)
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In Formulae I, Ia and Ib, m is preferably 1 or 2, and is most preferably 1.
Suitable ligands for use in the present invention which form metal complexes
resistant to
transchelation include: chelating agents, where 2-6, preferably 2-4, metal
donor atoms are
5 arranged such that 5- or 6-membered chelate rings result (by having a non-
coordinating
backbone of either carbon atoms or non-coordinating heteroatoms linking the
metal donor
atoms); or monodentate ligands which comprise donor atoms which bind strongly
to the
metal ion, such as isonitriles, phosphines or diazenides. Examples of donor
atom types
which bind well to metals as part of chelating agents are: amines, thiols,
amides, oximes
10 and phosphines. Phosphines form such strong metal complexes that even
monodentate or
bidentate phosphines form suitable metal complexes. The linear geometry of
isonitriles
and diazenides is such that they 'do not lend themselves readily to
incorporation into
chelating agents, and are hence typically used as monodentate ligands.
Examples of
suitable isonitriles include simple alkyl isonitriles such as tent-
butylisonitrile, and ether-
substituted isonitriles such as rnibi (i.e. 1-isocyano-2-methoxy-2-
methylpropane).
Examples of suitable phosphines include Tetrofosmin, and monodentate
phosphines such
as tris(3-methoxypropyl)phosphine. Examples of suitable diazenides include the
HYNIC
series of ligands i.e. hydrazine-substituted pyridines or nicotinamides.
2o Examples of suitable chelating agents for technetium which form metal
complexes
resistant to transchelation include, but are not limited to:
(i) diaminedioximes of formula:
~Q
Es NH
Ea Es
E ~~ N N ~ ~Es
OH OH
where EI-E6 are each independently an R' group;
each R' is H or Ci_lo alkyl, C3_io alkylaryl, C2_lo alkoxyalkyl, Cl_io
hydroxyalkyl, Ci_io
fluoroalkyl, CZ_io carboxyalkyl or CI_io aminoalkyl, or two or more R' groups
together
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I1
with the atoms to which they are attached form a carbocyclic, heterocyclic,
saturated or
unsaturated ring, and wherein one or more of the R' groups is conjugated to
the barbituric
acid MMP inhibitor;
and Q is a bridging group of formula -(J)r- ;
where f is 3, 4 or 5 and each J is independently -O-, NR'- or -C(R')a-
provided that -(J)r-
contains a maximum of one J group which is -O- or NR'-.
Preferred Q groups are as follows:
Q = -(CHZ)(CHR')(CH2)- ie. propyleneamine oxime or PnAO derivatives;
i o Q = -(CHZ)2(CHR')(CHZ)2- ie. pentyleneamine oxime or PentAO derivatives;
Q = -(CHa)ZNR'(CH2)a-.
EI to Eg are preferably chosen from: Cl_3 alkyl, alkylaryl alkoxyalkyl,
hydroxyalkyl,
fluoroalkyl, carboxyalkyl or aminoalkyl. Most preferably, each Ei to E6 group
is CH3.
The barbituric acid MMP inhibitor is preferably conjugated at either the Ei or
E6 R°
group, or an R' group of the Q moiety. Most preferably, the barbituric acid
MMP
inhibitor is conjugated to an R' group of the Q moiety. When the barbituric
acid MMP
inhibitor is conjugated to an R° group of the Q moiety, the R' group is
preferably at the
bridgehead position. In that case, Q is preferably -(CHZ)(CHR')(CHa)- ,
-(CH2)2(CHR')(CH2)2- or -(CH2)aNR°(CH2)a-, most preferably -
(CH2)a(CHR')(CH2)2-.
An especially preferred bifunctional diaminedioxime chelator has the Formula
III
(Chelator 1):
NH2
HN NH
\N N~
i I
OH OH
(III)
such that the synthetic barbituric acid MMP inhibitor is conjugated via the
bridgehead
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12
-CH2CHZNH2 group.
(ii) N3S ligands having a thioltriamide donor set such as MAG3
(mercaptoacetyltriglycine)
and related ligands; or having a diamidepyridinethiol donor set such as Pica;
(iii) N2S~ ligands having a diaminedithiol donor set such as BAT or ECD (i.e.
ethylcysteinate dimer), or an amideaminedithiol donor set such as MAMA;
(iv) N4 ligands which are open chain or macrocyclic ligands having a
tetramine,
l0 amidetriamine or diamidediamine donor set, such as cyclam, monoxocyclam or
dioxocyclam.
(v) Na02 ligands having a diaminediphenol donor set.
The above described ligands are particularly suitable for complexing
technetium eg. 94mTc
or ~9mTc, and are described more fully by Jurisson et al [Chem.Rev., 99, 2205-
2218
(1999)]. The ligands are also useful for other metals, such as copper (64Cu or
6~Cu),
vanadium (eg. 48~, iron (eg. SZFe), or cobalt (eg. SSCo). Other suitable
ligands are
described in Sandoz WO 91/01144, which includes ligands which are particularly
suitable
2o for indium, yttrium and gadolinium, especially macrocyclic aminocarboxylate
and
aminophosphonic acid ligands. Ligands which form non-ionic (i.e. neutral)
metal
complexes of gadolinium are known and are described in US 4885363. When the
radiometal ion is technetium, the ligand is preferably a chelating agent which
is
tetradentate. Preferred chelating agents for technetium are the
diaminedioximes, or those
having an N2Sa or N3S donor set as described above. Especially preferred
chelating
agents for technetium are the diaminedioximes:
It is strongly preferred that the synthetic barbituric acid matrix
metalloproteinase inhibitor
is bound to the metal complex in such a way that the linkage does not undergo
facile
metabolism in blood, since that would result in the metal complex being
cleaved off
before the labelled metalloproteinase inhibitor reached the desired in vivo
target site. The
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13
synthetic barbituric acid matrix metalloproteinase inhibitor is therefore
preferably
covalently bound to the metal complexes of the present invention via linkages
which are
not readily metabolised.
When the imaging moiety is a radioactive halogen, such as iodine, the
barbituric acid
MMP inhibitor is suitably chosen to include: a non-radioactive halogen atom
such as an
aryl iodide or bromide (to permit radioiodine exchange); an activated aryl
ring (e.g. a
phenol group); an organometallic precursor compound (eg. trialkyltin or
trialkylsilyl); or
an organic precursor such as triazenes. Methods of introducing radioactive
halogens
(including lz3I and isF~ are described by Bolton [J.Lab.Comp.Radiopharm., 45,
485-528
(2002)]. Examples of suitable aryl groups to which radioactive halogens,
especially
iodine can be attached are given below:
SnBu3
OH
Both contain substituents which permit facile radioiodine substitution onto
the aromatic
ring. Alternative substituents containing radioactive iodine can be
synthesised by direct
iodination via radiohalogen exchange, e.g.
127 ,~", 123'- ~ ~ 123' + 127'_
When the imaging moiety is a radioactive isotope of iodine the radioiodine
atom is
2o preferably attached via a direct covalent bond to an aromatic ring such as
a benzene ring,
or a vinyl group since it is known that iodine atoms bound to saturated
aliphatic systems
are prone to in vivo metabolism and hence loss of the radioiodine.
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14
When the imaging moiety comprises a radioactive isotope of fluorine (eg. 18F),
the
radioiodine atom may be carried out via direct labelling using the reaction of
I8F-fluoride
with a suitable precursor having a good leaving group, such as an alkyl
bromide, alkyl
mesylate or alkyl tosylate. 1gF can also be introduced by N-alkylation of
amine
precursors with alkylating agents such as 18F(CH~)30Ms (where Ms is mesylate)
to give
N-(CH2)318F, or O-alkylation of hydroxyl groups with 18F(CH2)30Ms or
18F(CH2)3Br.
For aryl systems, 18F-fluoride displacement of nitrogen from an aryl diazoniuW
salt is a
good route to aryl-~gF derivatives. See Bolton, J.Lab.Comp.Radiopharm., 45,
485-528
(2002) for a description of routes to 18F-labelled derivatives.
to
Preferred synthetic barbituric acid matrix metalloproteinase inhibitors of the
present
invention are of Formula IV:
O
HN_ _NH
O ~ 'O
(
where:
RI is R" or a Z group;
Ra is R", Y or NR4R5, where R4 is H or an R" group, R$ is H, C2_i4 acyl,
C2_io aminoalkyl or (N-C2_14 acyl)CZ_lo aminoalkyl
or an R" group, or R4 and RS together with the N atom to which they are
attached form an optionally (N-Ca_Ia)acylated Ca_g cycloarninoalkylene
ring;
R" is independently Ci_i4 alkyl, C3_$ cycloalkyl, Ca_14 alkenyl, C1_ia
fluoroalkyl,
Ci_i4 perfluoroalkyl, C6_i4 aryl, C2_~4 heteroaryl or C~_16 alkylaryl;
Z is a group of formula Al0[A20]pR3 where p is 0 or 1, and A1 and A2 are
independently Cl_io alkylene, C3_8 cycloalkylene, Cl_io perfluoroalkylene,
C6_IO arylene or CZ_lo heteroarylene, and R3 is an R group where R is
independently chosen from H, CI~ alkyl, C2.~ alkenyl, C2~ alkynyl,
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IS
Cl~ alkoxyalkyl or Ci~. hydroxyalkyl;
Y is a group of formula:
-N
where E is CR2, O, S or NR6; and R6 is CZ_14 acyl or an R" or Z group.
In Formula IV, R2 is preferably Y or NR4R5. When the imaging agent comprises a
barbituric acid MMP inhibitor of Formula IV, and the imaging moiety is a gamma-
emitting radioactive halogen or a positron-emitting radioactive non-metal, the
imaging
moiety may be attached at either of the RI or RZ substituents. When the
imaging moiety
is a radioactive or paramagnetic metal ion, the Ra substituent of Formula IV
is preferably
attached to or comprises the imaging moiety.
Especially preferred synthetic barbituric acid matrix metalloproteinase
inhibitors of the
present invention are of Formula V:
O
HN_ 'NH
O ~ 'O
N
~E
I s (v)
where E is CHR or NR6 and Rl is C6_ia n-alkyl, or C6_i4 aryl. Preferred
synthetic
barbituric acid matrix metalloproteinase inhibitors of Formula V are those
having E =
NR6 and R6 = C2_,4 acyl; -(CHZ)dOH, where d is 2, 3, 4 or 5; or -C6H4X where X
is H, Cl.
4 alkyl, Hal, OR, NR2, NOZ or S02NR~R8, where R' and R8 are independently R
groups,
and R is as defined in Formula IV (above).
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16
Especially preferred synthetic barbituric acid matrix metalloproteinase
inhibitors of
Formula V are those where RI is n-octyl, n-decyl, biphenyl, C6HSX or -C6H4-O-
C6H4X
where X is as defined above.
The barbituric acid MMP inhibitor compounds of the present invention are
prepared by
condensation of urea with mono- or di-substituted malonic ester derivatives.
Further
details are described by Foley et al [Bioorg.Med.Chem.Lett, 11, 969-972
(2001)]. The
MMP inhibitor compounds of Formula V can be prepared by the method of Grams et
al
[Biol.Chem., 382, 1277-1285 (2001)].
When the imaging agent of the present invention comprises a radioactive or
paramagnetic
metal ion, the metal ion is suitably present as a metal complex. Such metal
complexes
are suitably prepared by reaction of the conjugate of Formula Ib with the
appropriate
metal ion. The ligand-conjugate or chelator-conjugate of the barbituric acid
MMP
inhibitor of Formula Ib can be prepared via the bifunctional chelate approach.
Thus, it is
well known to prepare ligands or chelating agents which have attached thereto
a
functional group ("bifunctional linkers" or "bifunctional chelates"
respectively).
Functional groups that have been attached include: amine, thiocyanate,
maleimide and
active esters such as N-hydroxysuccinimide or pentafluorophenol. Chelator 1 of
the
2o present invention is an example of an amine-functionaIised bifunctional
chelate. Such
bifunctional chelates can be reacted with suitable functional groups on the
barbituric acid
matrix metalloproteinase inhibitor to form the desired conjugate. Such
suitable
functional groups on the barbituric acid include:
carboxyls (for amide bond formation with an amine-functionalised bifunctional
chelator);
amines (for amide bond formation with an carboxyl- or active ester-
functionalised
bifunctional chelator);
halogens, mesylates and tosylates (for N-alkylation of an amine-functionalised
bifunctional chelator) and
thiols (for reaction with a maleimide-functionalised bifunctional chelator).
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17
The radiolabelling of the especially preferred barbiturate MMP inhibitors of
the present
invention can be conveniently carried out using "precursors". When the imaging
moiety
comprises a metal ion, such precursors suitably comprise "conjugates" of the
barbiturate
MMP inhibitor with a ligand, as described in the fourth embodiment below. When
the
imaging moiety comprises a non-metallic radioisotope, ie. a gamma-emitting
radioactive
halogen or a positron-emitting radioactive non-metal, such "precursors"
suitably
comprise a non-radioactive material which is designed so that chemical
reaction with a
convenient chemical form of the desired non-metallic radioisotope can be
conducted in
the minimum number of steps (ideally a single step), and without the need for
significant
l0 purification (ideally no further purification) to give the desired
radioactive product. Such
precursors can conveniently be obtained in good chemical purity and,
optionally supplied
in sterile form.
It is envisaged that "precursors" (including ligand conjugates) for
radiolabelling of the
especially preferred barbiturate MMP inhibitors of the present invention can
be prepared
as follows:
where g = 2 or 3
Formula VI.
The terminal -OH group of the compound of Formula VI may be converted to a
tosyl or
mesyl group or bromo derivative, which can then be used to conjugate an amino-
functionalised chelator (shown in Scheme 1 for g = 2):
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18
Scheme 1.
HN NH HN NH
O~ ~O O 'O
Tosylate N
~N ~ \ or brominate
->
NJ ~ Chelate N
HO~ O ~ HN~ O
Chelate \
Br Br
Compound 2
The tosylate, mesylate or bromo groups of the precursors described may
alternatively be
displaced~with [18F]fluoride to give an 18F-labelled PET imaging agent.
Radioiodine derivatives can be prepared from the corresponding phenol
precursors:
O / I O ( ~ N O / I OH
O~ ~ ~ OH O
H O~~ H O
_OH N~OH
An alternative approach would be to use Compound 23 [Grams et al, Biol.Chem.,
382,
1277-1285 (2001) and Example 5 step (h)] for N-alkylation of an amine-
functionalised
chelator:
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19
Scheme 2.
HN NH HN NH
OBr 'O O ~O
I \ Chelate HN I \
O ~H HN O
\ I SH HS \ I
Br Br
Compound 23
Compound 23 can also be reacted with amines to give precursors suitable for
radioiodination, such as:
o i
O~ Br
N
O H ~ / SnBu3
The non-radioactive iodinated analogue Compound 24 has been prepared:
o
o / \
\I I~
O~ \ Br
N
OH ~ / I
Compound 24
Compound 23 can also be converted to an aryl trimethylsilyl (TMS) precursor
for
radioiodination:
N O / I O I \ N O / I O I \
\ / Br O~ \ / Br
H O~~ H O
N N
tms i
precursor iodinated derivative
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Compound 23 can be converted to an aryl diazonium precursor for
radiofluorination as
shown in Scheme 6:
Scheme 6
o ~ °~ o ~ o~ o
O~ \ ' I ~ Br O~ ~ I I ~ Br O~ O ~ I I ~ Br
H O ~~ -~. H O ~~ -~ '''1H O ~~
/ \ l \ / \
NOz r NHz +
Nz
Compound 19 ,$F
Br
Another approach would be to employ an amino group at the C-5 position. In
this way it
is expected that a chelator could be conjugated via a linker (Scheme 3):
10 Scheme 3
O O O
HN- _NH O O
or
O
H2N ~ ' ~ O Chelate
O
O or gr\ ~
_OMe
r
Br Br
Such primary amine substituted barbiturates can be prepared by alkylation of
Compound
23 with benzylamine, followed by removal of the benzyl protecting group under
standard
conditions such as hydrogenation using a palladium catalyst on charcoal.
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21
Another approach would be to use the piperazine derivative (Compound 6,
Example 7) to
attach a chelate. This could be via direct conjugation of the piperazine
substituent
secondary amine with a carboxyl- or active ester-functionalised bifunctional
chelator, or
via a linker. The latter is illustrated in Scheme 4, where an amine-
functionalised chelator
would be attached to the pendant carboxyl function of the linker:
Scheme 4
0 o 0
HN' -NH O O
or
O 'O
~N ~ ~ O O Chelate
HNJ ~ O
O or gr~ O
OMe I
Br
Compound 6
to Compound 6 can be acylated to give precursors suitable for radioiodination:
N O / I O I \ N O / I O I \
O~ \ / Br O~ \ / Br
H O~~ H O
N O N O
I I
\ SnBu3 \ i
precursor iodinated derivative
Compound 6 can also be reacted with a alkylating agent suitable for i$F
labelling such as
~8F(CH2)20Ts (where Ts is a tosylate group) or 18F(CH2)20Ms (where Ms is a
mesylate
group), to give the corresponding N-functionalised piperazine derivative
having an
N(CH2)218F substituent. Alternatively, Compound 6 can first be reacted with
chloroacetyl
chloride to give the N(CO)CH2Cl N-derivatised piperazine (Compound 11),
followed by
2o reaction with HS(CHa)318F:
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22
Scheme 5
H O / O \ HS~~eF
N \ I I /
O~ Br
N N
N- ~
H
CI
O O
Corr~pound 11
When the imaging agent of the present invention comprises a radioactive or
paramagnetic
metal ion, the metal ion is suitably present as a metal complex. Such metal
complexes
are suitably prepared by reaction of the conjugate of Formula Ib with the
appropriate
metal ion. The ligand-conjugate or chelator-conjugate of the barbituric acid
MMP
inhibitor of Formula Ib can be prepared via the bifunctional chelate approach.
Thus, it is
well known to prepare ligands or chelating agents which have attached thereto
a
functional group ("bifunctional linkers" or "bifunctional chelates"
respectively).
Functional groups that have been attached include: amine, thiocyanate,
maleimide and
active esters such as N-hydroxysuccinimide or pentafluorophenol. Chelator 1 of
the
present invention is an example of an amine-functionalised bifunctional
chelate. Such
bifunctional chelates can be reacted with suitable functional groups on the
barbituric acid
matrix metalloproteinase inhibitor to form the desired conjugate. Such
suitable
functional groups on the barbituric acid include:
carboxyls (for amide bond formation with an amine-functionalised bifunctional
chelator);
amines (for amide bond formation with an carboxyl- or active ester-
functionalised
bifunctional chelator);
halogens, mesylates and tosylates (for N-alkylation of an amine-functionalised
bifunctional chelator) and
thiols (for reaction with a maleimide-functionalised bifuncdonal chelator).
The radiometal complexes of the present invention may be prepared by reacting
a
solution of the radiometal in the appropriate oxidation state with the ligand
conjugate of
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23
Formula Ia at the appropriate pH. The solution may preferably contain a ligand
which
complexes weakly to the metal (such as gluconate or citrate) i.e. the
radiometal complex
is prepared by ligand exchange or transchelation. Such conditions are useful
to suppress
undesirable side reactions such as hydrolysis of the metal ion. When the
radiometal ion
is 99mTc, the usual starting material is sodium pertechnetate from a 99Mo
generator.
Technetium is present in 99mTc-pertechnetate in the Tc(VII) oxidation state,
which is
relatively unreactive. The preparation of technetium complexes of lower
oxidation state
Tc(I~ to Tc(V) therefore usually requires the addition of a suitable
pharmaceutically
acceptable reducing agent such as sodium dithionite, sodium bisulphite,
ascorbic acid,
formamidine sulphinic acid, stannous ion, Fe(II) or Cu(I), to facilitate
complexation. The
pharmaceutically acceptable reducing agent is preferably a stannous salt, most
preferably
stannous chloride, stannous fluoride or stannous tartrate.
When the imaging moiety is a hyperpolarised NMR-active nucleus, such as a
hyperpoIarised 13C atom, the desired hyperpolarised compound can be prepared
by
polarisation exchange from a hyperpolarised gas (such as lz9Xe or 3He) to a
suitable 13C-
enriched barbituric acid derivative.
In a second aspect, the present invention provides a pharmaceutical
composition which
2o comprises the imaging agent as described above, together with a
biocompatible carrier, in
a form suitable for mammalian administration. The "biocompatible carrier" is a
fluid,
especially a liquid, which in which the imaging agent can be suspended or
dissolved,
such that the composition is physiologically tolerable, ie. can be
administered to the
mammalian body without toxicity or undue discomfort. The biocompatible carrier
is
suitably an injectable carrier liquid such as sterile, pyrogen-free water for
injection; an
aqueous solution such as saline (which may advantageously be balanced so that
the final
product for injection is either isotonic or not hypotonic); an aqueous
solution of one or
more tonicity-adjusting substances (eg. salts of plasma cations with
biocompatible
counterions), sugars (e.g. glucose or sucrose), sugar alcohols (eg. sorbitol
or mannitol),
glycols (eg. glycerol), or other non-ionic polyol materials (eg.
polyethyleneglycols,
propylene glycols and the like).
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24
In a third aspect, the present invention provides a radiopharrnaceutical
composition which
comprises the imaging agent as described above wherein the imaging moiety is
radioactive, together with a biocompatible carrier (as defined above), in a
form suitable
for mammalian administration. Such radiopharmaceuticals are suitably supplied
in either
a container which is provided with a seal which is suitable for single or
multiple
puncturing with a hypodermic needle (e.g. a crimped-on septum seal closure)
whilst
maintaining sterile integrity. Such containers may contain single or multiple
patient
doses. Preferred multiple dose containers comprise a single bulk vial (e.g. of
10 to 30
cm3 volume) which contains multiple patient doses, whereby single patient
doses can thus
to be withdrawn into clinical grade syringes at various time intervals during
the viable
lifetime of the preparation to suit the clinical situation. Pre-filled
syringes are designed to
contain a single human dose, and are therefore preferably a disposable or
other syringe
suitable for clinical use. The pre-filled syringe may optionally be provided
with a syringe
shield to protect the operator from radioactive dose. Suitable such
radiopharmaceutical
syringe shields are known in the art and preferably comprise either lead or
tungsten.
When the imaging moiety comprises 9smTc, a radioactivity content suitable for
a
diagnostic imaging radiopharmaceutical is in the range 180 to 1500 MBq of
99mTc,
depending on the site to be imaged ih vivo, the uptake and the target to
background ratio.
In a fourth aspect, the present invention provides a conjugate of a synthetic
barbituric
acid matrix metalloproteinase inhibitor with a ligand, wherein the barbituric
acid
comprises a 5-position substituent, and said S-position substituent comprises
a ligand.
Said ligand conjugates are useful for the preparation of synthetic barbituric
acid matrix
metalloproteinase inhibitor labelled with either a radioactive metal ion or
paramagnetic
metal ion. Preferably, the ligand conjugate is of Formula Ib, as defined
above. Most
preferably, the synthetic barbituric acid MMP inhibitor of the ligand
conjugate is of
Formula IV, as defined above. Ideally, the synthetic barbituric acid MMP
inhibitor of the
ligand conjugate is of Formula V, as defined above. The ligand of the
conjugate of the
3o fourth aspect of the invention is preferably a chelating agent. Preferably,
the chelating
agent has a diaminedioxime, N2S2, or N3S donor set.
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In a fifth aspect, the present invention provides precursors useful in the
preparation of
radiopharmaceutical preparations where the imaging moiety comprises a non-
metallic
radioisotope, ie. a gamma-emitting radioactive halogen or a positron-emitting
radioactive
non-metal. Such "precursors" suitably comprise a non-radioactive derivative of
the
5 synthetic barbiturate matrix metalloproteinase inhibitor material which is
designed so that
chemical reaction with a convenient chemical form of the desired non-metallic
radioisotope can be conducted in the minimum number of steps (ideally a single
step), and
without the need for significant purification (ideally no further
purification) to give the
desired radioactive product. Such precursors can conveniently be obtained in
good
10 chemical purity. Suitable precursor derivatives are described in general
terms by Bolton,
J.Lab.Comp.Radiopharm., 45 485-528 (2002).
Preferred precursors of this embodiment comprise a derivative which either
undergoes
electrophilic or nucleophilic halogenation; undergoes facile alkylation with
an alkylating
15 agent chosen from an alkyl or fluoroalkyl halide, tosylate, triflate (ie.
trifluoromethanesulphonate) or mesylate; or alkylates thiol moieties to form
thioether
linkages. Examples of the first category are:
(a) organometallic derivatives such as a trialkylstannane (eg.
trimethylstannyl or
tributylstannyl), or a trialkylsilane (eg. trimethylsilyl);
20 (b) alkyl or aryl iodides or bromides for halogen exchange, and alkyl
tosylates or
mesylates for nucleophilic halogenation;
(c) aromatic rings activated towards electrophilic halogenation (eg. phenols)
and
aromatic rings activated towards nucleophilic halogenation (eg. aryl iodonium,
aryl diazonium or nitroaryl compounds).
Preferred derivatives which undergo facile alkylation are alcohols, phenols or
amine
groups, especially phenols and sterically-unhindered primary or secondary
amines.
Preferred derivatives which alkylate thiol-containing radioisotope reactants
are N-
haloacetyl groups, especially N-chloroacetyl and N-bromoacetyl derivatives.
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26
Preferred convenient chemical forms of the desired non-metallic radioisotope
include:
(a) halide ions (eg. I23I-iodide or I$F-fluoride), especially in aqueous
media, for
substitution reactions;
(b) I1C-methyl iodide or 18F-fluoroalkylene compounds functionalised with a
good leaving group, such as bromide, mesylate or tosylate;
(c) HS(CH2)318F for S-alkylation reactions with alkylating precursors such as
N-
chloroacetyl or N-bromoacetyl derivatives.
Examples of suitable such "precursors", and methods for their preparation are
described
in the first embodiment (above).
In a sixth aspect, the present invention provides a non-radioactive kit for
the preparation
of radioactive metal ion radiopharmaceutical compositions described above,
which
comprises a conjugate of a ligand with a synthetic barbituric acid matrix
metalloproteinase inhibitor. The ligand conjugates, and preferred aspects
thereof, are
described in the fourth embodiment above. Such kits are designed to give
sterile
radiopharmaceutical products suitable for human administration, e.g. via
direct injection
into the bloodstream. The kit is preferably lyophilised and is designed to be
reconstituted
with a convenient sterile source of the radiometal [eg. 99mTc-pertechnetate
(Tc04~ from a
99mTc radioisotope generator], to give a solution suitable for human
administration
2o without further manipulation. Suitable kits comprise a container (eg. a
septum-sealed
vial) containing the ligand or chelator conjugate in either free base or acid
salt form.
Alternatively, the kit may optionally contain a metal complex which, upon
addition of the
radiometal, undergoes transmetallation (i.e. metal exchange) giving the
desired product.
When the radioactive metal ion is 99mTc, the kit preferably further comprises
a
biocompatible reluctant, such as sodium dithionite, sodium bisulphate,
ascorbic acid,
formamidine sulphinic acid, stannous ion, Fe(II) or Cu(n. The biocompatible
reluctant is
preferably a stannous salt such as stannous chloride or stannous tartrate.
3o The non-radioactive kits may optionally further comprise additional
components such as a
transchelator, radioprotectant, antimicrobial preservative, pH-adjusting agent
or filler.
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27
The "transchelator" is a compound which reacts rapidly to form a weak complex
with the
radiometal, then is displaced by the ligand of the "conjugate". This minimises
the risk of
formation of radioactive impurities, eg. reduced hydrolysed technetium (RHT)
due to
rapid reduction of pertechnetate competing with technetium complexation.
Suitable such
transchelators are salts of a weak organic acid, ie. an organic acid having a
pKa in the
range 3 to 7, with a biocompatible cation. Suitable such weak organic acids
are acetic
acid, citric acid, tartaric acid, gluconic acid, glucoheptonic acid, benzoic
acid, phenols or
phosphonic acids. Hence, suitable salts are acetates, citrates, tartrates,
gluconates,
glucoheptonates, benzoates, phenolates or phosphonates. Preferred such salts
are
tartrates, gluconates, glucoheptonates, benzoates, or phosphonates, most
preferably
phosphonates, most especially diphosphonates. A preferred such transchelator
is a salt of
MDP, ie. methylenediphosphonic acid, with a biocompatible cation.
By the term "radioprotectant" is meant a compound which inhibits degradation
reactions,
such as redox processes, by trapping highly-reactive free radicals, such as
oxygen-
containing free radicals arising from the radiolysis of water. The
radioprotectants of the
present invention are suitably chosen from: ascorbic acid, par~a-aminobenzoic
acid (ie. 4-
aminobenzoic acid), gentisic acid (ie. 2,5-dihydroxybenzoic acid) and salts
thereof with a
biocompatible cation as described above.
By the term "antimicrobial preservative" is meant an agent which inhibits the
growth of
potentially harmful micro-organisms such as bacteria, yeasts or moulds. The
antimicrobial preservative may also exhibit some bactericidal properties,
depending on
the dose. The main role of the antimicrobial preservatives) of the present
invention is to
inhibit the growth of any such micro-organism in the radiopharmaceutical
composition
post-reconstitution, ie. in the radioactive diagnostic product itself. The
antimicrobial
preservative may, however, also optionally be used to inhibit the growth of
potentially
harmful micro-organisms in one or more components of the non-radioactive kit
of the
present invention prior to reconstitution. Suitable antimicrobial
preservatives) include:
the parabens, ie. methyl, ethyl, propyl or butyl paraben or mixtures thereof;
benzyl
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28
alcohol; phenol; cresol; cetrimide and thiomersal. Preferred antimicrobial
preservatives)
are the parabens.
The term "pH-adjusting agent" means a compound or mixture of compounds useful
to
ensure that the pH of the reconstituted kit is within acceptable limits
(approximately pH
4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting
agents
include pharmaceutically acceptable buffers, such as tricine, phosphate or
TRIS [ie.
tris(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such
as
sodium carbonate, sodium bicarbonate or mixtures thereof. When the conjugate
is
to employed in acid salt form, the pH adjusting agent may optionally be
provided in a
separate vial or container, so that the user of the kit can adjust the pH as
part of a multi-
step procedure.
By the term "filler" is meant a pharmaceutically acceptable bulking agent
which may
facilitate material handling during production and lyophilisation. Suitable
fillers include
inorganic salts such as sodium chloride, and water soluble sugars or sugar
alcohols such
as sucrose, maltose, mannitol or trehalose.
In a seventh aspect, the present invention provides kits for the preparation
of
radiopharmaceutical preparations where the imaging moiety comprises a non-
metallic
radioisotope, ie. a gamma-emitting radioactive halogen or a positron-emitting
radioactive
non-metal. Such kits comprise the "precursor" of the fifth embodiment,
preferably in
sterile non-pyrogenic form, so that reaction with a sterile source of the
radioisotope gives
the desired radiopharmaceutical with the minimum number of manipulations. Such
considerations are particularly important for radiopharmaceuticals where the
radioisotope
has a relatively short half life, and for ease of handling and hence reduced
radiation dose
for the radiopharmacist. Hence, the reaction medium for reconstitution of such
kits is
preferably aqueous, and in a form suitable for mammalian administration.
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The "precursor" of the kit is preferably supplied covalently attached to a
solid support
matrix. In that way, the desired radiopharmaceutical product forms in
solution, whereas
starting materials and impurities remain bound to the solid phase. Precursors
for solid
phase electrophilic fluorination with 18F-fluoride are described in WO
03/002489.
Precursors for solid phase nucleophilic fluorination with IgF-fluoride are
described in WO
03!002157. The kit may therefore contain a cartridge which can be plugged into
a
suitably adapted automated synthesizer. The cartridge may contain, apart from
the solid
support- bound precursor, a column to remove unwanted fluoride ion, and an
appropriate
vessel connected so as to allow the reaction mixture to be evaporated and
allow the
to product to be formulated as required. The reagents and solvents and other
consumables
required for the synthesis may also be included together with a compact disc
carrying the
software which allows the synthesiser to be operated in a way so as to meet
the customer
requirements for radioactive concentration, volumes, time of delivery etc.
Conveniently,
all components of the kit are disposable to minimise the possibility of
contamination
between runs and will be sterile and quality assured.
In an eighth aspect, the present invention discloses the use of the synthetic
barbituric acid
matrix metalloproteinase inhibitor imaging agent described above for the
diagnostic
imaging of atherosclerosis, especially unstable vulnerable plaques.
In a further aspect, the present invention discloses the use of the synthetic
barbituric acid
matrix metalloproteinase inhibitor imaging agent described above for the
diagnostic
imaging of other inflammatory diseases, cancer, or degenerative diseases.
In a further aspect, the present invention discloses the use of the synthetic
barbituric acid
matrix metalloproteinase inhibitor imaging agent described above for the
intravascular
detection of atherosclerosis, especially unstable vulnerable plaques, using
proximity
detection. Such proximity detection may be achieved using intravascular
devices such as
catheters or infra-operatively using hand-held detectors (eg. gamma
detectors). Such
intravascular detection is particularly useful when the imaging moiety is a
reporter group
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suitable for in vivo optical imaging or a ~3-emitter, since such moieties may
not be readily
detected outside the mammalian body, but are suitable for proximity detection.
5 The invention is illustrated by the non-limiting Examples detailed below.
Example 1
describes the synthesis of the compound 1,1,1-tris(2-aminoethyl)methane.
Example 2
provides an alternative synthesis of l,l,l-tris(2-aminoethyl)methane which
avoids the use
of potentially hazardous azide intermediates. Example 3 describes the
synthesis of a
chloronitrosoalkane precursor. Example 4 describes the synthesis of a
preferred amine-
10 substituted bifunctional diaminedioxime of the present invention (Chelator
1 ). Example
5 provides the synthesis of a non-radioactive iodinated barbiturate (Compound
4).
Example 6 describes the synthesis of the radioiodinated ~aSI analogue of
Compound 4
(Compound 5). Example 7 describes the synthesis of a piperazine-substituted
barbiturate
(Compound 6), where the piperazine amine can be used for further conjugation
(eg. of
15 chelating agents). Example 8 describes the synthesis of a fluoropropyl
derivative
(Compound 7), and Example 9 the corresponding 1gF analogue. Example 10
provides a
thioether-linked fluoropropyl derivative (Compound 9), and Example 11 the
corresponding 1gF derivative (Compound 10). Example 12 provides a synthesis of
a
chloroacetyl intermediate (Compound 11). Examples 13 and 14 provide the
syntheses of
20 chelator conjugates of the present invention (Compounds 16 and 17). Example
15
provides the synthesis of a tributylstannyl radioiodination precursor
(Compound 18).
Example 16 describes the synthesis of a bromoethyl derivative (Compound 13)
that acts
as a precursor for the radiosynthesis of the corresponding I8F analogue via
fluorodebromination with [1gF]fluoride. Example 17 provides the synthesis of
various
25 phenylpiperazine derivatives (Compounds 19 to 22). Example 18 describes the
synthesis
of Compound 24. Examples 19 and 20 describe ih vitro assays for assessing the
inhibitory activity of compounds of the invention vs specific
metalloproteinase enzymes.
Table 1 and Table 2 show the inhibition assay results for examples of non-
radioactive
iodinated, fluorinated and chelate derivatives of the invention with respect
to MMP-2,
30 MMP-9 and MMP-12. This shows that most compounds have similar inhibitory
activity
to that of the comparative prior art Compounds 2 and 3. This demonstrates that
a chelator
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31
or an imaging moiety such as an iodine atom or a fluorine atom can be
introduced
without compromising the biological activity of the barbiturate MMP inhibitor.
Example
21 describes the 9smTc-radiolabelling of chelator conjugates of the invention.
Example
22 describes a general method of radioiodination of suitable non-radioactive
precursors of
the invention.
Figure 1 shows the chemical structures of several compounds of the invention.
to Example 1: Synthesis of 1,1,1-tris(2-aminoethyl)methane.
Step 1(a): 3-(methoxycarbonylmethylene)~lutaric acid dimethylester.
Carbomethoxymethylenetriphenylphosphorane (167g, O.Smol) in toluene (600m1)
was
treated with dimethyl 3-oxoglutarate (87g, O.Smol) and the reaction heated to
100°C on
an oil bath at 120°C under an atmosphere of nitrogen for 36h. The
reaction was then
concentrated in vacuo and the oily residue triturated with 40/60 petrol
ether/diethylether
1:1, 600m1. Triphenylphosphine oxide precipitated out and the supernatant
liquid was
decanted/filtered off. The residue on evaporation in vacuo was Kugelrohr
distilled under
high vacuum Bpt (oven temperature 180-200°C at 0.2torr) to give
3-(methoxycarbonylmethylene)glutaric acid dimethylester (89.08g, 53%).
NMR 1H(CDC13): 8 3.31 (2H, s, CHZ), 3.7(9H, s, 3xOCH3), 3.87 (2H, s, CHI),
5.79 (1H,
s, =CH, ) ppm.
NMR 13C(CDC13), & 36.56,CH3, 48.7, 2xCH3, 52.09 and 52.5 (2xCH2); 122.3 and
146.16
C=CH; 165.9, 170.0 and 170.5 3xC00 ppm.
Step 1(b): Hydrogenation of 3-(methoxycarbonylmethylene)~lutaric acid
dimethylester.
3-(methoxycarbonylmethylene)glutaric acid dimethylester (89g, 267mmo1) in
methanol
(200m1) was shaken with (10% palladium on charcoal: 50% water) (9 g) under an
3o atmosphere of hydrogen gas (3.5 bar) for (30h). The solution was filtered
through
kieselguhr and concentrated in vacuo to give 3-(methoxycarbonylmethyl)glutaric
acid
dimethylester as an oil, yield (84.9g, 94 %).
NMR 1H(CDC13), 8 2.48 (6H, d, J=BHz, 3xCH2), 2.78 (1H, hextet, J--8Hz CH, )
3.7 (9H,
s, 3xCH3).
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NMR 13C(CDC13), ~ 28.6, CH; 37.50, 3xCH3; 51.6, 3xCH2; 172.28,3xC00.
Step 1(c): Reduction and esterification of trimethyl ester to the triacetate.
Under an atmosphere of nitrogen in a 3 necked 2L round bottomed flask lithium
aluminium hydride (20g, 588mmo1) in tetrahydrofuran (400m1) was treated
cautiously
with tris(methyloxycarbonylmethyl)methane (40g, 212mmol) in tetrahydrofuran
(200m1)
over lh. A strongly exothermic reaction occurred, causing the solvent to
reflux strongly.
The reaction was heated on an oil bath at 90°C at reflux for 3 days.
The reaction was
quenched by the cautious dropwise addition of acetic acid (100m1) until the
evolution of
hydrogen ceased. The stirred reaction mixture was cautiously treated with
acetic
anhydride solution (500mI) at such a rate as to cause gentle reflux. The flask
was
equipped for distillation and stirred and then heating at 90°C (oil
bath temperature) to
distil out the tetrahydrofuran. A further portion of acetic anhydride (300m1)
was added,
the reaction returned to reflux configuration and stirred and heated in an oil
bath at 140°C
for 5h. The reaction was allowed to cool and filtered. The aluminium oxide
precipitate
was washed with ethyl acetate and the combined filtrates concentrated on a
rotary
evaporator at a water bath temperature of 50°C in vacuo (5 mmHg) to
afford an oil. The
oil was taken up in ethyl acetate (500m1) and washed with saturated aqueous
potassium
carbonate solution. The ethyl acetate solution was separated, dried over
sodium sulphate,
and concentrated in vacuo to afford an oil. The oil was Kugelrohr distilled in
high
vacuum to give tris(2-acetoxyethyl)methane (45.3g, 96%) as an oil. Bp. 220
°C at 0.1
mmHg.
NMR 1H(CDCl3), 8 1.66(7H, m, 3xCH2, CH), 2.08(1H, s, 3xCH3); 4.1(6H, t,
3xCH2O).
NMR 13C(CDCl3), S 20.9, CH3; 29.34, CH; 32.17, CHa; 62.15, CH20; 171, CO.
Step 1(d): Removal of Acetate ~rouus from the triacetate.
Tris(2-acetoxyethyl)methane (45.3g, 165m1V1) in methanol (200m1) and 880
ammonia
(100m1) was heated on an oil bath at 80°C for 2 days. The reaction was
treated with a
further portion of.880 ammonia (50m1) and heated at 80°C in an oil bath
for 24h. A
further portion of 880 ammonia (50m1) was added and the reaction heated at
80°C for
24h. The reaction was then concentrated in vacuo to remove all solvents to
give an oil.
This was taken up into 880 ammonia (150m1) and heated at 80°C for 24h.
The reaction
was then concentrated in vacuo to remove all solvents to give an oil.
Kugelrohr
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distillation gave acetamide by 170-180 0.2mm. The bulbs containing the
acetamide were
washed clean and the distillation continued. Tris(2-hydroxyethyl)methane
(22.53g, 92%)
distilled at by 220 °C 0.2mm.
NMR IH(CDC13), ~ 1.45(6H, q, 3xCH2), 2.2(1H, quintet, CH); 3.7(6H, t 3xCH20H);
5.5(3H, brs, 3xOH).
NMR 13C(CDCl3), 8 22.13, CH; 33.95, 3xCH2; 57.8, 3xCH20H.
Step 1(e): Conversion of the trio! to the tris(methanesulphonate).
To an stirred ice-cooled solution of tris(2-hydroxyethyl)methane (lOg,
0.0676mo1) in
dichloromethane (SOmI) was slowly dripped a solution of methanesulphonyl
chloride
(40g, 0.349mo1) in dichloromethane (SOml) under nitrogen at such a rate that
the
temperature did not rise above 15°C. Pyridine (21.4g, 0.27mo1, 4eq)
dissolved in
dichloromethane (50m1) was then added drop-wise at such a rate that the
temperature did
not rise above 15°C, exothermic reaction. The reaction was left to stir
at room
temperature for 24h and then treated with SN hydrochloric acid solution (80m1)
and the
layers separated. The aqueous layer was extracted with further dichloromethane
(SOml)
and the organic extracts combined, dried over sodium sulphate, filtered and
concentrated
in vacu~ to give tris[2-(methylsulphonyloxy)ethyl]methane contaminated with
excess
methanesulphonyl chloride. The theoretical yield was 25.8g.
2o NMR 1H(CDC13), 8 4.3 (6H, t, 2xCH2), 3.0 (9H, s, 3xCH3), 2 (1H, hextet,
CH), 1.85 (6H,
q, 3xCH2).
Sten 1(f): Preparation of 1,1,1-tris(2-azidoethyl)methane.
A stirred solution of tris[2-(methylsulphonyloxy)ethyl]methane [from Step
1(e),
contaminated with excess methylsulphonyl chloride] (25.8g, 67mmol,
theoretical) in dry
DMF (250m1) under nitrogen was treated with sodium azide (30.7g, 0.47mo1)
porrion-
wise over 15 minutes. An exotherm was observed and the reaction was cooled on
an ice
bath. After 30 minutes, the reaction mixture was heated on an oil bath at
SO°C for 24h.
The reaction became brown in colour. The reaction was allowed to cool, treated
with
3o dilute potassium carbonate solution (200m1) and extracted three times with
40160 petrol
ether/diethylether 10:1 (3x150m1). The organic extracts were washed with water
(2x150m1), dried over sodium sulphate and filtered. Ethanol (200m1) was added
to the
petrol/ether solution to keep the triazide in solution and the volume reduced
in vacuo to
no less than 200m1. Ethanol (200m1). was added and reconcentrated in vacuo to
remove
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34
the last traces of petrol leaving no less than 200m1 of ethanolic solution.
The ethanol
solution of triazide was used directly in Step 1(g).
CARE: DO NOT REMOVE ALL THE SOLVENT AS THE AZIDE IS POTENTIALLY
EXPLOSIVE AND SHOULD BE KEPT 1N DILUTE SOLUTION AT ALL TIMES.
Less than 0.2m1 of the solution was evaporated in vacuum to remove the ethanol
and an
NMR run on this small sample:
NMR iH(CDCl3), 8 3.35 (6H, t, 3xCH2), 1.8 (1H, septet, CH, ), 1.6 (6H, q,
3xCH2).
Steu 1(~): Preuaration of 1,1,1-tris(2-aminoethyl)methane.
to Tris(2-azidoethyl)methane (15.06g, 0.0676 mol), (assuming 100% yield from
previous
reaction) in ethanol (200m1) was treated with 10% palladium on charcoal (2g,
50% water)
and hydrogenated for 12h. The reaction vessel was evacuated every 2 hours to
remove
0
nitrogen evolved from the reaction and refilled with hydrogen. A sample was
taken for
NMR analysis to confirm complete conversion of the triazide to the triamine.
15 Caution: unreduced azide could explode on distillation. The reaction was
filtered
through a Celite pad to remove the catalyst and concentrated in vacuo to give
tris(2-
aminoethyl)methane as an oil. This was further purified by Kugelrohr
distillation
bp.180-200°C at 0.4mm/Hg to give a colourless oil (8.1g, 82.7% overall
yield from the
triol).
20 NMR 1H(CDCI3), 2.72 (6H, t, 3xCH2N), 1.41 (H, septet, CH), 1.39 (6H, q,
3xCH2).
NMR 13C(CDCI3), 8 39.8 (CHZNHZ), 38.2 (CHa.), 31.0 (CH).
Examule 2: Alternative Preparation of 1,1,1-tris(2-aminoethyl)methane.
Steu 2(a): Amidation of trimethylester withp-methoxy-benzylamine.
Tris(methyloxycarbonylmethyl)methane [2 g, 8.4 mmol; prepared as in Step 1 (b)
above]
was dissolved in p-methoxy-benzylamine (25 g, 178.6 mmol). The apparatus was
set up
for distillation and heated to 120 °C for 24 hrs under nitrogen flow.
The progress of the
reaction was monitored by the amount of methanol collected. The reaction
mixture was
cooled to ambient temperature and 30 ml of ethyl acetate was added, then the
precipitated
triamide product stirred for 30 min. The triamide was isolated by filtration
and the filter
cake washed several times with sufficient amounts of ethyl acetate to remove
excessp-
. methoxy-benzylamine. After drying 4.6 g, 100 %, of a white powder was
obtained. The
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highly insoluble product was used directly in the next step without further
purification or
characterisation.
5 Step 2(b): Preparation of 1,1,1-tris[2-(p-methoxybenzylamino)ethyllmethane.
To a 1000 ml 3-necked round bottomed flask cooled in a ice-water bath the
triamide from
step 2(a) (10 g, 17.89 mmol) is carefully added to 250 ml of 1M borane
solution (3.5 g,
244.3 mmol) borane. After complete addition the ice-water bath is removed and
the
reaction mixture slowly heated to 60 °C. The reaction mixture is
stirred at 60 °C for 20
10 hrs. A sample of the reaction mixture (1 ml) was withdrawn, and mixed with
0.5 ml SN
HCl and left standing for 30 min. To the sample 0.5 ml of 50 NaOH was added,
followed
by 2 ml of water and the solution was stirred until all of the white
precipitate dissolved.
The solution was extracted with ether (5 ml) and evaporated. The residue was
dissolved
in acetonitrile at a concentration of 1 mg/ml and analysed by MS. If mono- and
diamide
15 (M+Hlz = 520 and 534) are seen in the MS spectrum, the reaction is not
complete. To
complete the reaction, a further 100 ml of 1M borane THF solution is added and
the
reaction mixture stirred for 6 more hrs at 60 °C and a new sample
withdrawn following
the previous sampling procedure. Further addition of the 1M borane in THF
solution is
continued as necessary until there is complete conversion to the triamine.
The reaction mixture is cooled to ambient temperature and SN HCl is slowly
added,
[CARE: vigorous foam formation occurs!]. HCl was added until no more gas
evolution
is observed. The mixture was stirred for 30 min and then evaporated. The cake
was
suspended in aqueous NaOH solution (20-40 %; 1:2 w/v) and stirred for 30
minutes. The
mixture was then diluted with water (3 volumes). The mixture was then
extracted with
diethylether (2 x 150 ml) (CARE: do not use halogenated solvents]. The
combined
organic phases were then washed with water (lx 200 ml), brine (150 ml) and
dried over
magnesium sulphate. Yield after evaporation: 7.6 g, 84 % as oil.
NMR 1H(CDC13), S: 1.45, (6H, m, 3xCHz; 1.54, (1H, septet, CH); 2.60 (6H, t,
3xCH~N); 3.68 (6H, s, ArCH2); 3.78 (9H, s, 3xCH30); 6.94(6H, d, 6xAr).
7.20(6H, d,
6xAr).
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NMR 13C(CDC13), 8: 32.17,CH; 34.44, CH2; 47.00, CHZ; 53.56, ArCH2; 55.25,
CH30;
113.78, Ar; 129.29, Ar; 132.61; Ar; 158.60, Ar;
Step 2(c): Preparation of 1,1,1-tris(2-aminoethyl)methane.
l,l,I-tris[2-(p-methoxybenzylamino)ethyl]methane (20.0 gram, 0.036 mol) was
dissolved in methanol (100 ml) and Pd(OH)Z (5.0 gram) was added. The mixture
was
hydrogenated (3 bar, 100 °C, in an autoclave) and stirred for 5 hours.
Pd(OH)a was
added in two more portions (2 x 5gram) after 10 and 15 hours respectively.
The reaction mixture was filtered and the filtrate was washed with methanol.
The
combined organic phase was evaporated and the residue was distilled under
vacuum
(1 x IO -2, 110 °C) to give 2.60 gram (50 %) of 1,1,1-ty-is(2-
aminoethyl)methane identical
with the previously described Example 1.
Example 3: Preparation of 3-chloro-3-methyl-2-nitrosobutane.
A mixture of 2-methylbut-2-ene (147m1, l.4mol) and isoamyl nitrite (156m1,
1.16mo1)
was cooled to -30 °C in a bath of cardice and methanol and vigorously
stirred with an
overhead air stirrer and treated dropwise with concentrated hydrochloric acid
(140m1,
1.68mo1 ) at such a rate that the temperature was maintained below -
20°C. This requires
2o about lh as there is a significant exotherm and care must be taken to
prevent overheating.
Ethanol (100m1) was added to reduce the viscosity of the slurry that had
formed at the end
of the addition and the reaction stirred at -20 to -10°C for a further
2h to complete the
reaction. The precipitate was collected by filtration under vacuum and washed
with
4x30m1 of cold (-20°C) ethanol and 100m1 of ice cold water, and dried
i~z vacuo to give 3-
chloro-3-methyl-2-nitrosobutane as a white solid. The ethanol filtrate and
washings were
combined and diluted with water (200m1) and cooled and allowed to stand for lh
at -10°C
when a further crop of 3-chloro-3-methyl-2-nitrosobutane crystallised out. The
precipitate
was collected by filtration and washed with the minimum of water and dried in
vacuo to
give a total yield of 3-chloro-3-methyl-2-nitrosobutane (115g 0.85mo1, 73%)
>98% pure
3o by NMR.
NMR 1H(CDCl3), As a mixture of isomers (isomerl, 90%) 1.5 d, (2H, CH3), 1.65
d,
(4H, 2 xCH3), 5.85,q, and 5.95,q, together 1H. (isomer2, 10%), 1.76 s, (6H, 2x
CH3),
2.07(3H, CH3).
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Example 4: Synthesis of bislN-(1,1-dimethyl-2-N-hydroxyimine propyl)2-
aminoethyll-(2-aminoeth~)methane (Chelator 1).
To a solution of tris(2-aminoethyl)methane (4.047g, 27.9mmo1) in dry ethanol
(30m1)
was added potassium carbonate anhydrous (7.7g, 55.8mmol, 2eq) at room
temperature
with vigorous stirnng under a nitrogen atmosphere. A solution of 3-chloro-3-
methyl-2
nitrosobutane (7.568, 55.8mo1, 2eq) was dissolved in dry ethanol (100m1) and
75m1 of
this solution was dripped slowly into the reaction mixture. The reaction Was
followed by
TLC on silica [plates run in dichloromethane, methanol, concentrated (0.88sg)
ammonia;
100/30/5 and the TLC plate developed by spraying with ninhydrin and heating].
The
1 o mono-, di- and tri-alkylated products were seen with RF's increasing in
that order.
Analytical HPLC was run using RPR reverse phase column in a gradient of 7.5-
75%
acetonitrile in 3% aqueous ammonia. The reaction was concentrated in vacuo to
remove
the ethanol and resuspended in water (1 l Oml). The aqueous slurry was
extracted with
ether (100m1) to remove some of the trialkylated compound and lipophilic
impurities
leaving the mono and desired dialkylated product in the water layer. The
aqueous
solution was buffered with ammonium acetate (2eq, 4.3g, 55.8mmo1) to ensure
good
chromatography. The aqueous solution was stored at 4°C overnight before
purifying by
automated preparative HPLC.
Yield (2.2g, 6.4mmol, 23 %).
2o Mass spec; Positive ion 10 V cone voltage. Found: 344; calculated M+H= 344.
NMR 1H(CDC13), ~ 1.24(6H, s, 2xCH3), 1.3(6H, s, 2xCH3), 1.25-1.75(7H, m,
3xCHa,CH), (3H, s, 2xCH2), 2.58 (4H, m, CHZN), 2.88(2H, t CHZN2), 5.0 (6H, s,
NHS ,
2xNH, 2xOH).
NMR 1H ((CD3)2S0) 81.1 4xCH; 1.29, 3xCHa; 2.1 (4H, t, 2xCH2);
NMR 13C((CD3)ZSO), S 9.0 (4xCH3), 25.8 (2xCH3), 31.0 2xCH2, 34.6 CHa, 56.8
2xCHaN;
160.3, C=N.
HPLC conditions: flow rate 8m1/min using a 25mm PRP column
A=3% ammonia solution (sp.gr = 0.88) lwater; B = Acetonitrile
3o Time %B
0 7.5
15 75.0
20 75.0
22 7.5
30 7.5
Load 3ml of aqueous solution per run, and collect in a time window of 12.5-
13.5 min.
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Example 5: Synthesis of Non-radioactive Iodine Barbiturate (Compound 4).
Step (a): 1-[4-(4-Iodo-phenoxy)phenyl]ethanone.
4-Fluoroacetophenone 25.0 g (181 mmol) was dissolved in DMF (180 ml), then 4-
Iodophenol (39.8 g, 181 mmol) and potassium carbonate (30.0 g, 217 mmol) were
added.
The mixture was refluxed for approximately 7 h, allowed to cool to room
temperature and
diluted with water. After extraction with methylene chloride or chloroform (3
times), the
combined organic phases were washed once with water, and dried (Na2S04). The
solvent
was removed in vacuo to provide the crude product. The brownish oily residue
was
recrystallised from hexanelethyl acetate (7:3) to yield the pure product as a
beige
l0 crystalline solid, 48.8 g (80%), mp: 99-101°C.
Step b: Preparation of 2-[4-(4-Iodo-phenoxy)phenyl]'-1-morpholin-4-yl-
ethanethione.
A mixture of 1-[4-(4-Iodo-phenoxy)phenyl]ethanone (23.0 g, 68.0 mmol), sulphur
(5.45
g, 170 mmol) and morpholine (11.8 g, 135 mmol) was heated at 150°C for
2.5 h. After
cooling in an ice bath, the mixture was treated with ethanol for a period of
30-60 min.
The precipitated bright yellow solid was collected by suction filtration and
recrystallised
from ethanol. The product contained a certain amount of sulphur. Yield 26.3 g
(88%) of
a mustard yellow solid. mp: 123-127°C.
Step (c): [4-(4-Iodo-phenoxy)phenyl]-acetic acid.
2-[4-(4-Iodo-phenoxy)phenyl]-1-morpholin-4-yl-ethanethione (26.9 g, 61.1 mmol)
was
heated together with a mixture of glacial acetic acid (54 ml), water (12 ml)
and cone.
sulphuric acid (8 ml) at 150°C for 12 h. After cooling to RT, the
reaction mixture was
diluted with water (ca. 10 mUmmol) and extracted with ethyl acetate (3x). The
combined
organic extracts were washed with water, dried (Na2S04) and the solvent
evaporated in
vacuo giving a beige solid (20.1 g, 93%). Mp: 148-150°C.
Step (d): [4-(4-Iodo-phenoxy)-phenyll-acetic acid methyl ester.
A solution of 17.3 g (48.9 mmol) [4-(4-Iodo-phenoxy)phenyl]-acetic acid in
methanol
(125 ml) was cooled to -10°C. Thionyl chloride (11.6 g, 7.1 ml, 97.8
mmol) was then
added and the reaction mixture heated to reflux for 1 h. After concentration
the residue
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was dissolved in ether. The ether phase was washed with water, dried (Na2S04)
and the
solvent evaporated to yield a viscous brown-red oil (13.6 g, 76%).
1H-NMR (300 MHz, CDCl3, TMS intern): 8 [ppm]: 7.49 (d, 3J= 8.9 Hz, 2H, H~,l),
7.15 (d, 3J= 8.9 Hz, 2H, H~.yl), 6.84 (d, 3J= 8.9 Hz, 2H, HAD,1), 6.66 (d, 3J=
8.9 Hz,
2H, Hp,D,I), 3.59 (s, 3H, COOCH3), 3.50 (s, 2H, CHz).
Step (e): 4-(4-Iodo-phenoxy)phenyll-malonic acid dimethyl ester.
A suspension of NaH (680 mg, 28.3 mmol) and dimethyl carbonate (8.16 g, 90.6
mmol)
in abs. dioxane (70m1) was heated to 100-120°C, then a solution of [4-
(4-Iodo-phenoxy)-
to phenyl]-acetic acid methyl ester (5.21 g, 14.2 mmol) in abs. dioxane (30
ml) was added
dropwise over a period of 1 h. Refluxing was continued for 3 h, then the
reaction mixture
was allowed to cool to RT overnight. The mixture was poured onto ice water and
subsequently extracted with methylene chloride (3x). The combined organic
phases were
washed with water (lx), brine (lx) , dried (NaaS04) and concentrated to give a
viscous
15 brown-red oil (5.25 g, 87%).
1H-NMR (400 MHz, CDCl3, TMS intern): 8 [ppm]: 7.53 (d, 3J= 8.7 Hz, 2H, H~,1),
7.29 (d, 3J= 8.7 Hz, 2H, H~,1), 6.89 (d, 3J= 8.7 Hz, 2H, HAzyl), 6.70 (d, 3J=
8.7 Hz,
2H, H~,l), 4.71 (s, 1H, CH), 3.68 (s, 6H, COOCH3).
20 Step (f~[4~4-Iodo-phenoxy)phenyl)-pyrimidine-2,4,6-trione.
Sodium (2 equivalents) was dissolved in ethanol (ca. 10 ml/mg), and urea (I.7
eq.) added
to the solution. A solution of 2-[4-(4-Iodo-phenoxy)-phenyl]-malonic acid
dimethyl ester
(2.22 g, 5.21 mmol) in ethanol was added dropwise, and the reaction mixture
heated to
reflux for 6 h. After cooling to RT, the mixture was poured onto ice water and
adjusted
25 to pH 2, using dilute hydrochloric acid. The precipitate was collected by
suction
filtration and dried in vacuo giving an amorphous solid. Recrystallisation
from
methanol/acetonitrile (1:1) gave a brown-yellow solid. Yield 480 mg (22%).
Mp: 285-286°C (decomposition).
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Step (g): 5-Bromo-5~4-~4-iodo-phenoxy)phen~]-pyrimidine-2,4,6-triune.
A suspension of 1.10 g (2.61 mmol) 5-[4-(4-Iodo-phenoxy)phenyl]-pyrimidine-
2,4,6-
triune, N-bromosuccinimide (557 mg, 3.31 mmol) and a catalytic amount of
dibenzoylperoxide (77 mg) in carbon tetrachloride (50 ml) was refluxed for 3
h. After
cooling to RT the mixture was concentrated, the residue treated with water and
then
extracted with ethyl acetate (3x). The combined extracts were washed with
brine, dried
(Na2S04) and the solvent evaporated giving a viscous brown oil, which was used
in the
next step without further purification (1.26 g, 96%).
10 Step (h): 5-[4-(2-Hydroxyethyl)piperazin-1-~]-5-[4-(4-iodo-phenoxy~phenv~'-
pyrimidine-2,4,6-triune (Compound 4~
A solution of 5-Bromo-5-[4-(4-iodo-phenoxy)phenyl]-pyrimidine-2,4,6-triune
(100 mg,
200 ~.mol) in methanol (5 ml) was treated with N-(2-Hydroxyethyl)piperazine
52.0 mg
(400 ~.mol) and the mixture stirred for 24 h at RT. A precipitate formed after
15 approximately 30-60 min, which was finally collected by suction and dried
ih vacuo,
giving the product as a colourless solid (73.0 mg, 67%).
mp: 255-257°C.
1H-NMR (300 MHz, DMSO-D6): 8 [ppm]: 11.78 (broad s, 2H), 7.93 (broad, d, 3J=
8.9
Hz, 2H, H~,l), 7.63 (broad, d, 3J= 8.9 Hz, 2H, H~.yl), 7.26 (broad, d, 3J= 8.9
Hz, 2H,
20 H~,l), 7.09 (broad, d, 3J= 8.9 Hz, 2H, HAryl), 4.53 (broad, s, 1H, OH),
3.70-3.66 (m,
2H, CHa), 2.80-2.58 (m, lOH, CHZ).
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Compounds 2, ie. 5-[4-(4-bromo-phenoxy)phenyl]-5-[4-(2-hydroxyethyl)piperazin-
1-yl]-
pyrimidine-2,4,6-trione and Compound 3, ie. 5-[4-(2-hydroxyethyl)piperazin-1-
yl]-5-[4-
(4-methoxy-phenoxy)phenyl]-pyrimidine-2,4,6-trione were prepared in an
analogous
manner, by the method of Grams et al [Biol.Chem., 382, 1277-1285 (2001)]
starting from
Compound 23, ie. 5-bromo-5-[4-(4-bromo-phenoxy)phenyl]-pyrimidine-2,4,6-trione
and
5-bromo-5-[4-(4-methoxy-phenoxy)phenyl]-pyrimidine-2,4,6-trione respectively
Example 6: Synthesis of 5-[4-(2-Hvdroxvethvl)piperazin-1-vll-5-(4-(4-
(~125lliodo-
to phenoxylphenyll-pyrimidine-2,4,6-trione (Compound 5).
2,5-Dihydroxybenzoic acid (0.6 mg, 3.9 p,mol}, ascorbic acid (0.8 mg, 4.5
pmol), water
for injection (20 ~1) and 5 p,l (65.3 nmol) CuS045H20 solution (cons. = 3.26
g/1 in water
for injection) were added to a conical vial containing Compound 2 (50 p,l, 209
nmol; conc
= 2.10 g/1 EtOH).
The ice-cooled mixture was degassed for 10 min using a He-flow, then 4 p,l
[125I]NaI in
NaOH solution (10.39 MBq) were added and the mixture vortexed. The mixture was
heated to 116°C for 60 min. After cooling to room temperature it was
diluted with 50 p,l
water for injection. The solution was injected to the gradient HPLC-
chromatograph with
y- and IJV-detector and a Nucleosil 100 C-18 5 p. 250x4.6 mm2 column with a
corresponding 20x4.6 mm2 precolumn.
HPLC-conditions: eluent A: CH3CN l H20 / TFA 950/50/1
eluent B: CH3CN / H2O / TFA 50/950/1
gradient: eluent B from 92% to 50% over 45 min, then from
SO% to 92% over 10 min
Flow: 1.5 ml/min
254 nm
Rt(product-fraction): 32.80-33.90 min.
3o A part of this fraction (200 pl) was reinjected to the gradient HPLC using
the same
conditions (see above).
Rt(Compound 5): 33.08 min
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42
The quality-control of this product (HPLC, same conditions) did not show any
impurities
in the y-channel. In the ITV-channel a very slight impurity (31.33 min.) was
detected
probably caused by the precursor Compound. It is possible to remove the
impurity from
the fraction with a second HPLC-run.
The Rt parameters were established by adding an aliquot of the non-radioactive
iodine
reference standard (Compound 4) to a second quality-control injection.
Radiochemical yield: 20%
Example 7: 5-f4-(4-Eromo-phenoxyluhenyll-5-piuerazin-1-yl-pyrimidine-2,4,6-
trione (Comuound 61.
5-Bromo-5-[4-(4-bromo-phenoxy)phenyl]-pyrimidine-2,4,6-trione [Compound 23,
Example 5 step (h)] (200 mg, 440 pmol) was dissolved in abs. methanol (5 ml)
and
treated with piperazine (75.8 mg, 880 ~,mol). After ca. 10 min a colourless
precipitate
formed. The reaction mixture was stirred for 24 h at RT., then the precipitate
was
collected under suction, stirred for 1 h in methanol and dried in vacuo to
give 160 mg
(79%) of a colourless solid.
mp: 265-266°C (decomposition).
1H-NMR (300 MHz, DMSO-D6): ~ [ppm]: 7.34 (broad, d, 3J=8.7 Hz, 2H, Hplyl),
7.22
(broad, d, 3J=8.7 Hz, 2H, H~yl), 6.82 (broad, d, 3J=8.7 Hz, 2H, H~.yl), 6.79
(broad, d,
3J=8.7 Hz, 2H, H~,ryl), 2.55-2.23 (broad, m, 8H, CHa).
Example 8: 5-[4-(4-Bromophenoxy)phenyl~-5-(4-(3-fluoronrouyl)-piperazin-1-yl)-
uyrimidine-2,4,6-trione (Compound 7).
To a solution of Compound 6 (10 mg, 2.2 x 10-5 mol) in pyridine (2 ml) under a
nitrogen
atmosphere at room temperature was added 3-fluoropropyltosylate (l.l
equivalents). The
reaction was stirred for 16 hours. The mixture was concentrated and dissolved
in
3o methanol (5 ml). The mixture was purified by HPLC (C18, 150 x 10 mm) and
the product
eluted after circa 13 minutes. The solvent was removed to give an off white
solid (yield
38%). The structure was confirmed by mass spectral [ES (+ve) 521.1] and 1H NMR
analysis.
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a
[ 18F] F-/ Kr y p 2. 2. 2/ K2C03
18
TsO~OTs F OTs
CH3CN, 100°C/ 10 rri n
hia a two-way tap I~ryptofix 222 (lOmg) in acetonitrile (300 ~1) and potassium
carbonate
(4mg) in water (300 ~1), prepared in a glass vial, was transferred using a
plastic syringe
(lml) into a carbon glass reaction vessel sited in a brass heater. 18F-
fluoride (185-
370MBq) in the target water (0.5-Zml) was then added through the two-way tap.
The
heater was set at 125°C and the timer started. After l5mins three
aliquots of acetonitrile
(O.Sml) were added at lmin intervals. The 18F-fluoride was dried up to 40mins
in total.
After 40mins, the heater was cooled down with compressed air, the pot lid was
removed
and 1,3-propanediol-di p-tosylate (5-l2mg) and acetonitrile (lml) was added.
The pot lid
was replaced and the lines capped off with stoppers. The heater was set at
100°C and
labelled at 100°C/l0mins. After labelling, 3-[I$F] fluoropropyl
tosylate was isolated by
Gilson RP HPLC using the following conditions:
Column u-bondapak C18 7.8x300mm
Eluent Water (pump A): Acetonitrile
(pump B)
Loop Size lml
Pump speed 4ml/min
Wavelength 254nm
Gradient S-90% eluent B over 20 min
Product Rt 12 min
Once isolated, the cut sample (ca. lOml) was diluted with water (1 Oml) and
loaded onto a
conditioned C18 sep pak. The sep pak was dried with nitrogen for l5mins and
flushed
off with an organic solvent, pyridine (2ml), acetonitrile (2ml) or DMF (2m1).
Approx.
99% of the activity was flushed off.
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Sten (bl: alkvlation of Comuound 6.
0
O \ ~ O ~ ~ ,sF~OTs OJN O ~ ' ' ~ Br
O~ Br ~1
H O~~ H O
N N
H
~~$F
Compound 6 Compound 8
Compound 6 can be alkylated to give Compound 8 by refluxing in pyridine with 3-
[18F]
fluoropropyl tosylate.
Example 10: 5-f4-(4-Bromophenoxy)nhenyll-5-f4-(2-fluorouropylsulfanyl)acetyll-
piperazin-1-yl~-pyrimidine-2,4,6-trione (Compound 9).
Step (a): 3-firitylsulfanyl-propan-1-of [Ph~CS(CH2~OH]_
Triphenylmethanol (390.6 mg, 1.5 mmol) in TFA (10 ml) was added dropwise to a
stirred
solution of 3-mercaptopropan-1-of (129.6 pl, 1.5 mmol) in TFA (10 ml). After
the
addition TFA was evaporated under reduced pressure and the crude product
immediately
purified by reverse phase preparative chromatography (Phenomenex Luna C18
column,
OOG-4253-V0; solvents A= water / 0.1% TFA and B= CH3CN / 0.1% TFA; gradient 70-
80 % B over 60 min; flow 50 ml / minute; detection at 254 nm), affording 372
mg (74%)
of pure compound. (analytical HPLC: Vydac C 18 column, 218TP54: solvents: A=
water /
0.1% TFA and B= CH3CN / 0.1% TFA; gradient 70-80 % B over 20 min; flow 1.0 ml
/minute; retention time 5.4 minutes detected at 214 and 254 nm). Structure
verified by
2o NMR.
Step (b): Methanesulfonic acid 3-trit Is~ ulfan ~~l-propyl ester
[Ph~CS(CH~~OMsI.
To 3-Tritylsulfanyl-propan-I-of (372.0 mg, 1.I1 mmol) dissolved in THF_(10 ml)
was
added triethylamine (151.7 mg, 209 ~1, 1.5 mmol) and mesyl chloride (171.9 mg,
116.6
~ul, 1.5 mmol). After 1 hour the precipitate was filtered off, THF evaporated
under
reduced pressure and the crude product purified by reverse phase preparative
chromatography (Phenomenex Luna C18 column, OOG-4253-V0; solvents A= water /
O.I% TFA and B= CH3CN / 0.1% TFA; gradient 80-100 % B over 60 min; flow 50 ml
l
minute; detection at 254 nm}, affording 318 mg (69%) of pure compound.
(analytical
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HPLC: Vydac C18 column, 218TP54: solvents: A= water / 0.1 % TFA and B= CH3CN /
0.1% TFA; gradient 60-70 % B over 20 min; flow 1.0 ml /minute; retention time
18.7
minutes detected at 214 and 254 nm). Structure verified by NMR.
Step (cL(3-fluoropropylsulfanyl)triphenylmethane [Ph~CS CH~F]_
Potassium fluoride (1.4 mg, 0.024 mmol) and kryptofix 222 (9.0 mg, 0.024 mmol)
were
dissolved in acetonitrile (0.2 ml) (heating). Methanesulfonic acid 3-
tritylsulfanyl-propyl
ester (S mg, 0.012 mmol) in acetonitrile (0.2 ml) was added. The reaction
mixture was
heated to 80 °C for 90 minutes. The crude product was purified by
reverse phase
10 preparative chromatography (Vydac C18 column, 218TP1022; solvents A= water
/ 0.1%
TFA and B= CH3CN / 0.1 % TFA; gradient 40-90 % B over 40 min; flow 10 ml /
minute;
detection at 214 nm). A yield of 2.5 mg (62 %) of purified material was
obtained
(analytical HPLC: Phenomenex Luna C18 column, OOB-4251-E0: solvents: A= water
/
0.1 % TFA and B= CH3CN / 0.1 % TFA; gradient 40-80 % B over 10 min; flow 2.0
ml
15 /minute; retention time 8.2 minutes detected at 214 and 254 nm). Structure
verified by
NMR.
Step (d)y 5~4-(4-Bromophenoxy~phen~l-5-i4-(2-fluoropropylsulfanyl)acetyl]-
piperazin
1-yl~pyrimidine-2,4,6-trione (Compound 9~,
20 3-Fluoro-tritylsulfanyl-propane (4.1 mg, 0.021 mmol) was stirred with TFA
(100 pl),
triisopropylsilane (10 pl) and water (10 p,l). Water (300 ~.l) was added
followed by 200 p,l
potassium carbonate (aq). Compound 11 (3.25 mg, 0.0061 mmol) in CH3CN (500
~.l)
was added. The pH was adjusted to 10 with potassium carbonate (aq). The
mixture was
heated to 75°C for half an hour. The crude product was purified by
reverse phase
25 preparative chromatography (Phenomenex Luna C18 column, OOG-4253-N0;
solvents A=
water / 0.1% TFA and B= CH3CN / 0.1% TFA; gradient 20-70 % B over 30 min; flow
5
ml / minute; detection at 254 nm). A yield of 2 mg (55%) of purified material
was
obtained (analytical HPLC: Vydac C18 column, 218TP54: solvents: A= water /
0.1%
TFA and B= CH3CN / 0.1 % TFA; gradient 20-70 % B over 20 min; flow 1.0 ml
/minute;
30 retention time 17.4 minutes detected at 214 and 254 nm).
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46
IH NMR (CHC13-dl, TMS reference): 8 2.01 (m, 2H), 8 2.72 (broad t, 2H), 8 2.75
(t, 2
H), 8 2.79 (broad t, 2 H), 8 3.30 (s, 2 H), 8 3.30 (broad t, 2 H), 8 3.5I (s,
2 H), 8 3.64
(broad t, 2 H), 8 4.53 (dt, 2 H), 8 6.93 (complex d, 2 H), 8 6.99 (complex d,
2 H), S 6.93
(complex d, 2 H), 8 7.46 (complex d, 2 H), 8 7.48 (complex d, 2 H), & 7.77
(complex d,
2 H).
Example 11: 5-[4-(4-Sromophenoxyl-uhenyll-5-d4-(2-fl$Fl-fluorouro_pylsulfanyl)-
acetyll-pinerazin-1-yl)-uyrimidine-2,4,6-trione (Compound 10)
Step (a): Preparation of 3-[18F] fluoro-mt ls~nyl-pro ane
[~8F]F-/Kryp 2.2.2/K2C03
TrS~pMs TrS~~$p
DMSO, 80°C/5 min
Via a two-way tap Kryptofix 222 (lOmg) in acetonitrile (800 ~.l) and potassium
carbonate
(lmg) imwater (50 ~,l), prepared in a glass vial, was transferred using a
plastic syringe
(lml) to the carbon glass reaction vessel situated in the brass heater. ~$F-
fluoride (185-
370 MBq) in the target water (0.5-2ml) was then also added through the two-way
tap.
The heater was set at 125°C and the timer started. After 1 Smins three
aliquots of
acetonitrile (O.Sml) were added at lmin intervals. The 1$F-fluoride was dried
up to
40mins in total. After 40mins, the heater was cooled down with compressed air,
the pot
lid was removed and trimethyl-(3-tritylsulfanyl-propoxy)silane (1-2mg) and
DMSO
(0.2m1) was added. The pot lid was replaced and the lines capped off with
stoppers. The
heater was set at 80 °C and labelled at 80 °C/Smins. After
labelling, the reaction mixture
was analysed by RP HPLC using the following HPLC conditions:
Column u-bondapak C18 7.8x300mm
Eluent 0.1%TFA/Water (pump A): 0.1%TFA/Acetonitrile (pump B)
Loop Size 100u1
Pump speed 4m1/min
Wavelength 254nm
Gradient 1 wins 40%B
15 mins 40-80%B
5 wins 80%B
The reaction mixture was diluted with DMSO/water (1:1 v/v, O.lSml) and loaded
onto a
conditioned t-C18 sep-pak. The cartridge was washed with water (lOml), dried
with
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47
nitrogen and 3-[18F] fluoro-1-tritylsuIfanyl-propane was eluted with 4
aliquots of
acetonitrile (O.SmI per aliquot).
Step (b): Preparation of 3-[I$Fl fluoro-propane-1-thiol
TFA/ TI S/ Vlt~t er
TrS~~sF HSWsF
80°G 10 rri n
A solution of 3-[18F] fluoro-1-tritylsulfanyl-propane in acetonitrile (1-2 ml)
was
evaporated to dryness using a stream of nitrogen at 100°C/1 Omins. A
mixture of TFA
(O.OSmI), triisopropylsilane (O.Olml) and water (O.Olml) was added followed by
heating
to at 80°C/lOmins to produce 3-[18F] fluoro-propane-1-thiol.
Step (c): Reaction with Compound 11.
HS~',aF N o / ~
N~ / Br O~ ~ ~ Br
H O~' H
N O
~CI N~S~/'vaF
O IfO
Compound 11 Compound 10
A general procedure for labelling a chloroacetyl precursor is to cool the
reaction vessel
15 containing the 3-[18F] fluoro-1-mercapto-propane from Step (b) with
compressed air, and
then to add ammonia (27% in water, O.lml) and the Compound 11 precursor (lmg)
in
water (0.05m1). The mixture is heated at 80 °C/ lOmins.
2o Example 12: 5-f4-(4-Bromophenoxv)-uhenyll-5-f4-(2-chloroacetyl)-piperazin-1-
yl)-
~yrimidine-2,4,6-trione (Comuound 11).
To a flask charged with nitrogen and Compound 6 (50 mg, 1.1 x 10'~ mol) was
added
dichloromethane (15 ml). The reaction mixture was cooled in an ice/water bath.
Chloroacetyl chloride (14 p,1) and triethylamine (14 p,l) were added
sequentially. The ice
25 bath was removed after 10 minutes and the mixture was allowed to warm to
ambient
temperature. After 18 hours the sample was concentrated. Methanol (2 ml) was
added and
the mixture was separated by HPLC (C18, 150 x 10 mm). The product eluted at
17.5
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minutes (52% yield). The structure was confirmed by mass spectral [ES(-ve)
535.1) and
1H NMR analysis
Examine 13: 3-(4-f 5-(4-(4-Bromophenoxy)-phenyll-2,4,6-
trioxohexahydropyrimidin-5-yl~-piperazin-1-yl)-N-f 5-(2-hydroxynimino-1,1-
dimethyluropynamino)-3-(2-(2-hydroxylamino-1,1-dimethylpropylamino)-ethyll
pentyl~-propionamide (Compound 16).
Step (a): 5-(4-(4-Bromo-phenoxy)-phenyl]'-5-[4-(2-carboxyethyl)piperazin 111
pyrimidine-2,4,6-trione (Compound 1~
200 mg (440 ~Cmol) 5-Bromo-5-[4-(4-bromo-phenoxy)phenyl]-pyrimidine-2,4,6-
trione
(Compound 23, Example 5) was dissolved in 2 ml abs. methanol and treated with
83.5
mg (5.28 rnmol, 1.2 eq.) 3-(piperazin-lyl)propionic acid. The reaction mixture
was
heated to reflux for 6 h and then concentrated. The yellow, solid residue was
I5 recrystallised from water to give 170 mg (320 ,umol, 72%) of a colourless
amorphous
solid.
mp: 208-210°C (decomposition).
1H-NMR (300 MHz, DMSO-D6): 8 [ppm]: 7.64 (broad, d, 3J=8.6 Hz, 2H, HAiyl),
7.50
(broad, d, 3J=8.6 Hz, 2H, H~,1), 7.14 (broad, d, 3J=8.6 Hz, 2H, HAiyl), 7.10
(broad, d,
3J=8.6 Hz, 2H, H~,l), 2.75-2.39 (m, 12H, CHI).
Step f b)
To a solution of Compound 14 (53 mg) in N,N dimethylformamide (10 ml) under a
nitrogen atmosphere was added TBTU (85 mg) and N methylmorpholine (0.01 ml)
sequentially. After ten minutes Chelator 1 (35 mg) was added and the reaction
mixture
was stirred at room temperature for 24 hours. The solvent was removed at
reduced
pressure and the mixture was dissolved in methanol (5 ml). The crude mixture
was
separated by HPLC. The product eluted after circa 10 minutes (75% yield). The
structure
was confirmed by mass spectral [ES(+ve) 858.1] and IH NMR analysis.
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Example I4: Synthesis of Compound 17.
Step (a): 5-f4-(2-Aminoeth~) piperazin-1-yl]-5-[4-(4-bromo-
phenoxylphenyllpyrimidine-2 4 6-trione (Compound 12)
200 mg (440 p,mol) 5-Bromo-5-[4-(4-bromo-phenoxy)phenyl]pyrimidine-2,4,6-
trione
(Compound 23, Example 5) was dissolved in 2 ml abs. methanol and treated with
125 mg
(127 pl, 9.67 p.mol) N-(2-aminoethyl)piperazine. The reaction mixture was
stirred at RT,
and after ca. 30 min a colourless precipitate formed. Stirring was continued
for 16 h, then
the precipitate was collected by suction and dried in vacuo to give 100 mg
(200 ~,mol,
45%) of a colourless solid.
mp: 220-223°C (decomposition).
1H-NMR (300 MHz, DMSO-D6): 8 [ppm]: 7.67 (broad, d, 3J=9.0 Hz, 2H, HA~.yh
ortho
to C-Br), 7.55 (broad, d, 3J=9.0 Hz, 2H, H~.yI, ortho to Cq°art,
attached to Pyr.-C 5), 7.15
(broad, d, 3J=9.0 Hz, 2H, H~,1, meta to Cq"art, attached to Pyr.-C 5), 7.12
(broad, d,
3J=9.0 Hz, 2H, HAryl, meta to C-Br), 2.89-2.79 (m, 2H, CHI-NH2), 2.77-2.65 (m,
4H, N
1-CH2), 2.39-2.58 (m, 6H, N 4-CH2).
Step (b): 4-f2-(4-~5-f4-(4-Bromophenoxy) phenyl]-2 4 6-
trioxohexahydropyrimidin 5
yll-piperazin-1-yl)-ethylcarbamoyll-butyric acid Compound 15,~
To a solution of Compound 12 in N,N dimethylformamide (30 ml) under a nitrogen
atmosphere was added glutaric anhydride (11 mg) and triethylamine (0.01 ml)
sequentially. After 24 hours the solvent was removed under reduced pressure.
The crude
mixture was dissolved in methanol (5 ml) and separated by HPLC. The product
eluted
after 12 minutes (50% yield). The structure was confirmed by mass spectral
[ES(+ve)
617.9] and'H NMR analysis.
Step (c): Coniu~ation of 4-f2-(4-~5-f4-(4-bromophenoxy)-phenyll-2 4 6-
trioxohexahydropyrimidin-5-yll-piperazin-1-~ -ethylcarbamoyll-butyric acid
with
Chelator 1.
To a solution of Compound 15 (11 mg) in dichloromethane (5 ml) was added TBTU
(8
mg) and N methylmorpholine (0.1 ml) under a nitrogen atmosphere. After 5
minutes,
Chelator 1 (6 mg) was added and the mixture stirred for 24 hours. The solvent
was
removed at reduced pressure and the mixture was dissolved in methanol (5 mI).
The
mixture was separated by HPLC and the product eluted after circa 10 minutes
(58%
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yield). The structure was confirmed by mass spectral [ES(+ve) 943.2] and 1H
NMR
analysis.
Example 15: 5-f4-(2-Hydroxyethyl) piperazin-1-yll-5-[4-(4-
tributylstannylphenoxy)-
phenyll-pyrimidine-2,4,6-trione (Compound 18).
To a suspension of Compound 2 (80 mg) in toluene under a nitrogen atmosphere
was
added Pd(PPh3)4 (200 mg) and hexabutylditin (0.2 ml). The yellow mixture was
heated at
reflux for 24 hours. After this time the reaction mixture had become black in
colour. The
10 reaction mixture was filtered and the. solvent was removed at reduced
pressure. 'The crude
mixture was dissolved in methanol and purified by HPLC (yield 45%). The
structure was
confirmed by mass spectral [ES(+ve) 715.1) and IH NMR analysis.
15 Example 16: 5-[4-(2-Bromoethyl)piperazin-1-yll-5-f4-(4-bromo-
phenoxy)phenyll-
pyrimidine-2,4,6-trione (Compound 13).
To a suspension of Compound 2 (1.40 g, 2.78 mmol) in 80 ml acetonitrile was
added 1.46
g (5.56 mmol) triphenylphosphine and 1.84 g (5.56 mmol) carbon tetrabromide.
The
mixture was heated to reflux for a period of 18 h, cooled to RT and stored at -
30°C
20 overnight. The solid precipitate, which formed upon cooling was collected
by suction to
give 920 mg (58%) of a beige solid.
'H-NMR (300 MHz, DMSO-D6~: 8 [ppmJ: 7.56 (d, 3J=9.0 Hz, 2H, HAryl), 7.40 (d,
3J=8.7 Hz, 2H, HAryl), 7.09 (d, J=9.0 Hz, 2H, HAryl), 7.02 (d, 3J=8.7 Hz, 2H,
HAry1),
3.83-2.70 (m, 12H, CH2).
Example 17: Synthesis of Phenyl-Piperazine Derivatives (Compounds 19 to 22)
(a General procedure: Compounds 19 to 21
The corresponding phenyl piperazine (2.0 eq.) was added in portions to a
solution of
Compound 23 [Example 5 step (h)] (1.0 eq.) in abs. methanol (ca. 2-3 ml/mmol).
The
3o reaction mixture was stirred at RT for 20 h. The precipitate was collected
under suction
and washed with methanol.
In this manner were prepared:
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5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(4-nitrophenyl)piperazin-1-yl]-pyrimidine-
2,4, 6-
trione (Compound 19) - from the reaction of 400 mg (880 ~.mol) Compound 2 and
365
mg (1.76 mmol) 1-(4-nitrophenyl)piperazine in 4 ml methanol was obtained 320
mg
(63%) of the bright yellow reaction product after 20 h.
1H-NMR (400 MHz, DMSO-D6): 8 [ppm]:8.22-7.04 (m, 12H, H,4,y~), 3.80-2.77 (m,
8H,
CHa).
5-[4-(4-Bromo-phenoxy)phenyl]-5-[4-(4-fluorophenyl)piperazin-1-y1]-pyrimidine-
2,4,6-
trione (Compound 20) - from the reaction of 400 mg (880 wmol) Compound 2 and
317
mg (1.76 mmol) 1-(4-fluorophenyl)piperazine in 2.5 ml methanol was obtained
after
recrystallisation from chloroform 290 mg (60%) of the colourless reaction
product,
mp: 247-249.5 °C.
1H-NMR (400 MHz, DMSO-d6): 8 [ppm]: 11.66 (s, 2H, NH), 7.59-6.92 (m, 12H,
H,~,~,~),
3.33-2.74 (m, 8H, CHZ).
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(4-trimethylsilyl-phenyl)piperazin-1-yl]-
pyrimidine-2,4,6-trione (Compound 21) - from the reaction of 400 mg (880
~,mol)
Compound 2 and 413 mg (1.76 mmol) 1-(4-trimethylsilylphenyl)piperazine
in 2.5 ml methanol was obtained 440 mg (82%) of the colourless reaction
product.
2o mp:205-210°C.
1H-NMR (400 MHz, DMSO-D6): 8 [ppm]:7.93-6.77 (m, 12H, H~y~), 3.64-2.66 (m, 8H,
CHz), 0.20 (s, 9H, SiCH3).
(b) 5-f4-(4-Bromo-phenoxy)-phenyl]-5-[4-(4-iodophenyl)piperazin-1-
~],=pyrimidine-
2,4,6-trione (Compound 22).
To a suspension of 280 mg (460 ~.mol) Compound 21 in 25 ml methanol was added
a
solution of 300 mg (1.84 mmol) iodine monochloride in 5 ml methanol over 40
min at -
70°C under an argon atmosphere. The orange solution was allowed to warm
to RT over a
period of 1.5 h, diluted with dichloromethane and washed until colourless with
10%
aqueous sodium thiosulfate. The water phase was extracted with dichloromethane
(3x),
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washed with brine and dried (Na2S04). The solvent was evaporated and the
residue dried
in uacuo to give 230 mg of the raw product.
Recrystallisation from methanol gave 62 mg (20%) of the colourless crystalline
product.
mp: 210-211 °C.
1H-NMR (300 MHz, DMSO-d6): 8 [ppm]: 11.55 (s, 2H, NH), 7.50-6.63 (m, 12H,
HAry~),
3.03 (s, 4H, CH2), 2.63 (s, 4H, CHa).
Example 18: 5-[4-(4-Bromophenoxy)-uhenyll-5-(4-iodophenylamino)-uyrimidine-
2,4,6-trione (Compound 24).
To a solution of Compound 23 (Example 5, 90 mg) in dichloromethane (20 ml) was
added 1.1 equivalents of 4-iodoaniline (50 mg) and triethylamine (0.2 ml). The
reaction
was stirred under a nitrogen atmosphere for 16 hours. The solvent was removed
at
reduced pressure. The residue was dissolved in methanol (2 ml). The crude
mixture was
separated by HPLC and the new compound eluted after circa 20.5 minutes. The
solvent
was removed at reduced pressure to yield an off white solid (85% yield). The
structure
was confirmed by mass spectral [ES(-ve) 591.9] and 1H NMR analysis.
Example 19: In Yitro Metallonroteinase inhibition assay.
Compounds 2 to 4 and 20 were studied by the method of Huang W. et al. [J Biol
Chem.
272, 22086-22091 (1997)].
Thus, a constant concentration of the fluorogenic substrate (1 ~,M) and the
respective
MMPs (1nM) were incubated with increasing amounts of the MMP-inhibitors (100pM-
100 ~M) to determine their ICso values. 'The results are shown in Table 1:
Table 1
Compound MMP-2 ICS (nM) MMP-9 ICso (nM)
2 (prior art) 9 4
3 (prior art) 9 6
4 7 2
20 12 19
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Example 20: Additional lh hitro Metallonroteinase inhibition assay.
Compounds were screened using the following commercially available Biomol
assay
kits:
MMP-2 colourimetric assay kit - Catalogue number AK-408,
MMP-9 colourimetric assay kit - Catalogue number AK-410,
MMP-12 colourimetric assay kit - Catalogue number AK-402,
Which are available from Affiniti Research Products Ltd. (Palatine House,
Matford
Court, Exeter, EX2 8NL, UK).
to (a) Test Compound Preparation.
Inhibitors were provided in powdered form, and stored at 4°C. For each
inhibitor a 1mM
stock solution in DMSO was prepared, dispensed into 20,1 aliquots and these
aliquots
stored at -20°C. The stock solution was diluted to give 8 inhibitor
concentrations
(recommended: SO~.M,~S~.M, SOOnM, SOnM, SnM, SOOpM, 50pM and SpM). Dilutions
were made in the kit assay buffer. A five-fold dilution of the inhibitor
stocks is made on
addition to the assay wells, therefore final concentration range is from lOpM
to lpM.
(bLperimental Procedure.
Details are provided with the commercial kit, but can be summarised as
follows:
- Prepare test compound dilutions as above,
- Add assay buffer to plate,
- Add test compounds to plate
- Prepare standard kit inhibitor NNGH (see kit for dilution factor)
- Add NNGH to control inhibitor wells
- Prepare MMP enzyme (see kit for dilution factor)
- Add MMP to plate
- Incubate plate at 37°C for ~l5min
- Prepare thiopeptolide substrate (see kit for dilution factor)
- Add substrate to plate
- Count every 2min for lhr, 37°C, 414nm on a Labsystems iEMS plate
reader.
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(c) Results.
The results are given in Table 2:
Table 2
Compound MMP-2 (Ki) MMP-9 (Ki) MMP-12 (Ki)
nM n~M~ nM
7 11 2 -
9 5 0.3 11
16 14 3 -
17 29 10 157
24 45 20 -
The yymTc complexes were prepared in the same manner, by adding the following
to an
nitrogen-purged P46 vial:
l0 1 ml N2 purged MeOH,
100~g of Compound 16 (or 17) in 100.1 MeOH,
O.SmI NaaC03lNaHC03 buffer (pH 9.2),
O.SmI Tc04 from Tc generator,
0.1 ml SnCla/lVmP solution,
(solution containing 10.2mg SnCl2 and lOlmg methylenediphosphonic
acid in 100m1 N2 purged saline).
For 99mTc-Compound 16 the activity of solution was measured to be 216 MBq and
the
solution was heated to 37°C for 33min. An ITLC (Instant thin layer
chromatography)
using SG plates and a mobile phase of MeOH/(NH40Ac O.1M) 1:1 showed 1% RHT
(reduced hydrolysed Tc) at the origin. HPLC analysis showed 93% of 9~"Tc-
Compound
16 to give an RCP of 92%.
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99mTc-Compound 17 was prepared in a similar manner. The activity of the
complex
solution was measured as 203 MBq. ITLC gave 4% colloid and HPLC analysis
showed
93% 99mTc-Compound 17 to give an RCP of 89%.
HPLC analyses were carried out using an Xterra RP18, 3.S~,m, 4.6 x 150 mm
column
using an aqueous mobile phase (solvent A) of 0.06% NH40H and organic mobile
phase
(solvent B) of acetonitrile and a flow rate of lml/min. Typical gradients used
were as
follows : 0-5 min (IO-30% B), 5-17 min (30% B), 17-18 min (30-100% B), 18-22
min
(100% B) and 22-24 min (100-10% B). The retention time of 99"'Tc-Compound 16
was
10 7.6 min while that of 99mTc- Compound 17 was 7.Smin.
Example 22: General Procedure for Electrouhilic Radioidination of Barbiturate
Precursors.
15 10~L of freshly prepared O.O1M peracetic acid in water (1 x 10-~ mol) is
added to a
silanised vial containing the precursor substrate (1 x 10'' mol) in an
appropriate solvent,
together with 200~,L 0.2M NH40Ac buffer (pH = 4), 100~.L NaI~~I (1 x 10-~ mol)
and
Na123I . The reaction is agitated gently and the product purified by HPLC.
